Process for preparing compounds having triple activities of thrombolysis, antithrombotic and radical scavenging

ABSTRACT

The present invention relates to a compound simultaneously having triple activities of thrombolysis, antithrombosis and free radical scavenging, as well as the preparation method, composition, and applications thereof. The compound is represented by the formula I shown below: 
     
       
         
         
             
             
         
       
     
     wherein the definitions of T, Q, R 1  and R 2  are described herein. The compound of the present invention simultaneously has triple functions of thrombolysis, free radical scavenging and thrombus-targeting/antithrombosis. The present invention also relates to a pharmaceutical composition comprising the compound, and a preparation method and a nanostructure of the compound.

This application is a divisional of U.S. application Ser. No.14/956,723, filed Dec. 2, 2015; which is a continuation ofPCT/CN2014/079098, filed Jun. 3, 2014; which claims the priority ofCN201310225330.6, filed Jun. 5, 2013. The contents of the aboveapplications are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence_Listing.txt with a creation date of Dec. 26, 2017, anda size of 1.60 kilobytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

TECHNICAL FIELD

The present invention relates to a novel compound simultaneously havingthrombolytic, free radical scavenging andthrombus-targeting/antithrombotic functions as well as preparationmethod and uses thereof. The present invention further relates to anovel binary conjugate formed by coupling a thrombolytic oligopeptideand a tetrahydroisoquinoline compound having two C₁₋₄ alkyl groups via alinking arm. The present invention also relates to a pharmaceuticalcomposition comprising the compound, a preparation method and ananostructure of the compound.

BACKGROUND

The incidence rate of thrombotic diseases, such as stroke/infarction,ranks first in a variety of diseases. There is an increasing trend inthe incidence rate of the diseases in recent years, and the diseasesbecome a serious threat to human health. Drug treatment of thethrombotic diseases is the focus and hotspot of treating thrombosis.Currently, there are many limitations on clinically applied thromboticdrugs, and searching for a safe and effective new thrombus drug is oneof the research hotspots.

According to present research, in addition to anti-platelet aggregationand anti-thrombotic activities,3S-1,1-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid also has free radical scavengingactivity. Moreover, in Chinese Patent Publication CN101497651B, filedJan. 30, 2008, 10 tetrahydroisoquinoline compounds having thrombolyticactivity were disclosed. These tetrahydroisoquinoline compounds include3S-6,7-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-acyl-Pro-Ala-Lys,3S-6,7-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-acyl-Arg-Pro-Ala-Lys,3S-6,7-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-acyl-Ala-Arg-Pro-Ala-Lys,3S-6,7-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-acyl-Gly-Arg-Pro-Ala-Lys,3S-6,7-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-acyl-Gln-Arg-Pro-Ala-Lys,3S-2-[Pro-Ala-Lys]-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-carboxylicacid,3S-2-[Arg-Pro-Ala-Lys]-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-carboxylicacid,3S-2-[Ala-Arg-Pro-Ala-Lys]-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-carboxylicacid,3S-2-[Gly-Arg-Pro-Ala-Lys]-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-carboxylicacid, and3S-2-[Gln-Arg-Pro-Ala-Lys]-1,2,3,4-tetrahydro-6,7-dihydroxy-isoquinoline-3-carboxylicacid. The compounds above were abbreviated as“6,7-dihydroxy-isoquinolines having thrombolytic activity.” However, theeffective dosages of these 6,7-dihydroxy-isoquinolines havingthrombolytic activity are higher, and the anti-thrombotic activity andthe free radical scavenging activity were not disclosed or verified.Furthermore, the efficacy in treating stroke was demonstrated to beeffective only at the time of stroke onset. As for treating strokebeyond 30 minutes from onset of syndrome, the efficacy was not disclosedor verified.

Therefore, for effectively and safely treating thrombotic diseases inclinical practice, a novel compound that simultaneously hasthrombolytic, anti-thrombotic, and free radical scavenging activities,may effectively cross the blood-brain barrier (BBB), and may achieve thedescribed effects at a low dose is needed.

SUMMARY

The first aspect of the present invention is to provide a compoundhaving formula I:

-   -   wherein T represents a linking arm having at least two groups        for linking, Q represents a peptide having thrombolytic        activity, R₁ and R₂ represents C₁₋₄ alkyl groups, wherein R₁ and        R₂ may be the same or different.

In one embodiment of the present invention, at least one of the groupfor linking of the linking arm T is an amino group, and the remaininggroups for linking are carboxyl group or amino group.

In a preferred embodiment of the present invention, the linking arm Tmay be a natural amino acid, such as L-Lys, L-Asp, or L-Glu.

In a more preferred embodiment of the present invention, the linking armmay be L-Lys.

In a preferred embodiment of the present invention, the peptide havingthrombotic activity used in the present invention is selected from agroup consisting of an oligopeptide having a PA (Pro-Ala) sequence, aPAK (Pro-Ala-Lys) sequence, a AKP (Ala-Lys-Pro) sequence or a KAP(Lys-Ala-Pro) sequence, and a peptide comprising repeated units of thePAK sequence, the AKP sequence or the KAP sequence.

In an embodiment of the present invention, the oligopeptide havingthrombolytic activity may be a tripeptide to octapeptide containing a PA(Pro-Ala) sequence, a PAK sequence, a AKP sequence or a KAP sequence,preferably a tripeptide containing the PA sequence. In a more preferredembodiment, the tripeptide has a chemical formula of Q1 or Q2 shownbelow:

Pro-Ala-AA  (Q1)

AA-Ala-Pro  (Q2)

wherein AA is selected from a group consisting of L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu.

In a preferred embodiment, R₁ and R₂ both are methyl group.

In a preferred embodiment, the R₁ and R₂ of the formula I are methylgroups, the linking arm is L-Lys, L-Asp, or L-Glu, and the peptidehaving thrombolytic activity is a tripeptide containing a PA (Pro-Ala)sequence. For example, the compound may have the formula Ia (such ascompound 5Aa-p in FIG. 1), Ib (such as compound 5Ba-p in FIG. 2), Ic(such as compound 5Ca-p in FIG. 3), Id (such as compound 5 Da-p in FIG.4), Ie (such as compound 5Ea-p in FIG. 5), If (such as compound 5Fa-p inFIG. 6), Ig (such as compound 5Ga-p in FIG. 7), or Ih (such as compound5Ha-p in FIG. 8):

wherein AA is selected from a group consisting of L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu.

The second aspect of the present invention is to provide apharmaceutical composition comprising the compound of the presentinvention described above and a pharmaceutically acceptable carrier.

In a preferred embodiment of the present invention, the compounds may bein the form of nanospherical structure.

In a preferred embodiment of the present invention, the pharmaceuticalcomposition may be used as a thrombolytic drug, a NO free radicalscavenging drug, or a thrombus-targeting/antithrombotic drug.

In another preferred embodiment of the present invention, thepharmaceutical composition may be used as a drug in treating stroke orcerebral infarction, more preferably, in treating stroke or cerebralinfarction beyond 4 hours, 6 hours, and 24 hours from the onset of thesyndrome, and treating via successive administration.

The third aspect of the present invention is to provide a preparationmethod of the compounds having formula I. The method comprises thefollowing steps:

(1) provide a compound having formula II:

-   -   wherein R1 and R2 are C1-4 alkyl groups, and R1 and R2 are the        same or different.

(2) providing the linking arm T having at least two groups for linking,and the peptide Q having thrombolytic activity, wherein the linking armhaving a first group for linking and a second group for linking.

(3) coupling the carboxyl group of the compound having formula II withthe first group for linking of the linking arm T to form a compoundhaving formula IM-1:

under appropriate reaction condition; and

(4) coupling the peptide Q having thrombolytic activity with thecompound having formula IM-1 under appropriate reaction condition,wherein one terminal of the peptide Q having thrombolytic activity iscoupled to the second group for linking of the linking arm T to form thecompound having formula I.

In an embodiment of the present invention, the first group for linkingof the linking arm T is an amino group being used to couple to thecarboxyl group of the compound of formula II in a condensation reaction.Moreover, the second group for linking is a carboxyl group or an aminogroup being used to couple to the N-terminal or C-terminal of thepeptide Q having thrombolytic activity. The definitions of the linkingarm T and the peptide Q having thrombolytic activity used in thepreparation method of the invention are the same as the definitions ofthe compound having formula I above.

In a preferred embodiment of the present invention, the linking arm inthe present preparation method may be L-Lys, L-Asp, or L-Glu, and morepreferably L-Lys. The peptide having thrombolytic activity may be atripeptide containing a PA (Pro-Ala) sequence, an oligopeptidecontaining a PAK (Pro-Ala-Lys) sequence, a AKP (Ala-Lys-Pro) sequence ora KAP (Lys-Ala-Pro) sequence, or a peptide having a repeated sequencecontaining the PAK sequence, the AKP sequence or the KAP sequence, andmore preferably a tripeptide having a formula of Q1 or Q2 shown below:

Pro-Ala-AA  (Q1)

AA-Ala-Pro  (Q2)

wherein AA is selected from a group consisting of L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu.

In another preferred embodiment of the present invention, the R₁ and R₂of the compound having formula II both are methyl groups, the linkingarm is L-Lys, L-Asp, or L-Glu, and the peptide having thrombolyticactivity is a tripeptide containing a PA (Pro-Ala) sequence. In a morepreferred embodiment of the present invention, the preparation method ofthe present invention may be used to form the compounds having the aboveformulas Ia-h.

The in vivo tests in rats showed that the compounds or thepharmaceutical compositions of the present invention have excellentthrombolytic and antithrombotic activities at low dose, and mayeffectively protect the neurological function of the stroke rats, thusmay effectively and safely treat thrombotic diseases in clinicalpractice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a synthesis scheme of compound Ia according to one embodimentof the present invention;

FIG. 2 is a synthesis scheme of compound Ib according to one embodimentof the present invention;

FIG. 3 is a synthesis scheme of compound Ic according to one embodimentof the present invention;

FIG. 4 is a synthesis scheme of compound Id according to one embodimentof the present invention;

FIG. 5 is a synthesis scheme of compound Ie according to one embodimentof the present invention;

FIG. 6 is a synthesis scheme of compound If according to one embodimentof the present invention;

FIG. 7 is a synthesis scheme of compound Ig according to one embodimentof the present invention;

FIG. 8 is a synthesis scheme of compound Ih according to one embodimentof the present invention; and

FIG. 9 shows TEM photographs of nanostructures of compounds 5Aa-paccording to one embodiment of the present invention.

DETAILED DESCRIPTION

The detailed description provided below in connection with the appendeddrawings and embodiments is used to illustrate the technical solution ofthe present invention. However, the scopes of the present inventionshould be regarded as including but not limited thereto.

In the present invention, the tetrahydroisoquinoline compounds havingtwo C₁₋₄ alkyl groups (i.e. the compound of formula II) and the peptidehaving thrombolytic activity are coupled via a linking arm to form anovel binary conjugate simultaneously having triple functions ofthrombolytic, free radical scavenging, andthrombus-targeting/antithrombotic activities. The conjugate above isabbreviated as “the novel binary conjugate of the present invention”below.

Since two C₁₋₄ alkyl groups were introduced in position 1 and a linkingarm was introduced in position 3 of the compound having formula II, “thenovel binary conjugate of the present invention” has the following fouradvantages, as compared with “the 6,7-dihydroxyisoquinoline havingthrombolytic activity.” 1) The steric hindrance effect of the two C₁₋₄alkyl groups introduced in position 1 of the compound having formula IImay block the approach of the carboxypeptidase and the aminopeptidase,thus the thrombolytic oligopeptide of “the novel binary conjugate of thepresent invention” will not be easily hydrolyzed; 2) the hydrophobiccontribution of the two C₁₋₄ alkyl groups introduced in position 1 ofthe compound having formula II may allow “the novel binary conjugate ofthe present invention” to cross the blood-brain barrier with betterefficiency; 3) the electron donating effect of the two C₁₋₄ alkyl groupsintroduced in the position 1 of the compound having formula II may allowthe reducing ability of “the novel binary conjugate of the presentinvention” to satisfy the need for free radical scavenging; 4) thelinking arm introduced in position 3 of the compound having formula IImay allow “the novel binary conjugate of the present invention” toaggregate effectively together to form nanospherical structures having adiameter of 20-210 nm, preferably in a range of 20-100 nm. This stablenanostructure may assist “the novel binary conjugate of the presentinvention” in not being engulfed by macrophage in blood circulation,thus may be safely transported toward the thrombus forming site andfinally cross the blood-brain barrier. In summary, “the novel binaryconjugate of the present invention” may form a nanostructure to achievethe function of crossing blood-brain barrier. In addition tothrombolytic and antithrombotic functions, “the novel binary conjugateof the present invention” may also effectively scavenge the freeradicals of OH, NO, and superoxide anions, and may achieve effectivethrombolysis at low dose, thereby giving good prospects for clinicalapplication.

As used herein, “group for linking” means a functional group, such as acarboxyl group or an amino group, capable of performing condensationreaction.

As used herein, “linking arm” means a molecule having the groups forlinking, capable of coupling the compound having formula II with theoilgopeptide Q having thrombolytic activity. At least one group forlinking of the linking arm is an amino group, and the remaining groupsfor linking are carboxyl groups or amino groups. According to theinvention, the linking arm may be a natural amino acid, such as L-Lys,L-Asp, or L-Glu.

The introduced linking arm allows “the novel binary conjugate of thepresent invention” to form a stable nanospherical structure not beingengulfed by macrophages. The nanospherical structure may be safelytransported toward the thrombus forming site and finally cross theblood-brain barrier. Especially, when the linking arm is L-Lys, “thenovel binary conjugate of the present invention” may effectivelyaggregate together to form nanospherical structures having a diameter of20-210 nm, preferably in a range of 20-100 nm. The stable nanostructuremay assist in preventing “the novel binary conjugate of the presentinvention” from being engulfed by macrophages in blood circulation, soas to enable its safe transportation toward the thrombus formation siteand, finally, crossing the blood-brain barrier.

As used herein, an “oligopeptide” means a small peptide molecule havinga molecular weight below 1000 Dalton (D) and usually consisting of 3 to8 amino acids.

As used herein, a “peptide having thrombolytic activity” means anoligopeptide thrombolytic agent having functions of increasing vascularpermeability and thrombolysis, including P6A (ARPAK) (SEQ ID NO: 1),metabolites of P6A, and related derivatives. A previous study hasdisclosed that Pro-Ala-Lys was the shortest sequence with good activity,and also the stablest sequence among several thrombolytic oligopeptides,including Ala-Arg-Pro-Ala-Lys (SEQ ID NO: 1), Gly-Arg-Pro-Ala-Lys (SEQID NO: 2), Gln-Arg-Arg-Pro-Ala-Lys (SEQ ID NO: 3) and Pro-Ala-Lys.Introducing a tripeptide having Pro-Ala-AA sequence to the position 3 ofthe compound having formula II via the linking arm may allow “the novelbinary conjugate of the present invention” to obtain better stabilityand stronger thrombolytic activity.

For example, an oligopeptide containing the sequence of PAK, AKP, or KAPused in the present invention may be PAK, RPAK (Arg-Pro-Ala-Lys) (SEQ IDNO: 4), ARPAK (Ala-Arg-Pro-Ala-Lys) (SEQ ID NO: 1), GRPAK(Gly-Arg-Pro-Ala-Lys) (SEQ ID NO: 2), QRPAK (Gln-Arg-Pro-Ala-Lys) (SEQID NO: 5), AKP, KAP, KPAK (Lys-Pro-Ala-Lys) (SEQ ID NO: 6), PAKP(Pro-Ala-Lys-Pro) (SEQ ID NO: 7), AKPAK (Ala-Lys-Pro-Ala-Lys) (SEQ IDNO: 8), or PAKPA (Pro-Ala-Lys-Pro-Ala) (SEQ ID NO: 9).

For example, the peptide having repeating units of the PAK sequence, theAKP sequence or the KAP sequence used in the present invention may beany of those peptides being described in the Chinese patent publicationCN101190941 as a peptide having thrombolytic activity, including apeptide having repeating units of the PAK sequence, such as (PAK)₂,(PAK)₃, (PAK)₄, (PAK)₅ and (PAK)₆; a peptide having repeating units ofthe AKP sequence, such as (AKP)₂, (AKP)₃, (AKP)₄, (AKP)₅ and (AKP)₆; anda peptide having repeating units of the KPA sequence, such as (KPA)₂,(KPA)₃, (KPA)₄, (KPA)₅ and (KPA)₆.

As used herein, “C₁₋₄ alkyl group” means an alkyl group having 1-4carbons, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, orsec-butyl, tert-butyl. When R₁ and R₂ of the compound having formula Iboth are methyl groups, the compound having formula II, used as astarting material, may be obtained by Pictet-Spengler condnesation of3,4-dihydroxy-L-phenylalanine and acetone in the presense oftrifluoroacetate (TFA) and anhydrous magnesium sulfate. The advantage iseasier preparation.

In the present invention, a pharmaceutical composition may be anyclinically acceptable and suitable formulations. Preferably, theformulation is injectable formulations (powder for injection,lyophilized powder for injection, liquid for injection, infusion, etc.).The pharmaceutically acceptable carrier may be mannitol, water, Ringer'ssolution, or isotonic sodium chloride solution, etc.

The nanospherical structure of the compounds according to the presentinvention has a diameter of 20-210 nm, and more preferably, 20-100 nm,by which the compounds may cross the blood-brain barrier moreefficiently.

The stable nanostructure may help the compounds of the present inventionnot to be engulfed by macrophages, by which the compounds may be safelytransported toward the thrombosis formation site and eventually crossthe blood-brain barrier. The pharmaceutical composition of the presentinvention may be used as a thrombolytic drugs in treating myocardialinfarction, ischemic stroke, deep vein thrombosis, pulmonary embolism,peripheral arterial occlusive disease, occluded central vascular accessdevices, clotted arteriovenous fistula and shunts, carotid stenosis,etc. The pharmaceutical composition of this invention may also be usedas a NO free radical scavenging drug in treating neurodegenerativediseases, cardiovascular disease, mental illness, altitude sickness,diabetes, rheumatoid arthritis, traumatic brain injury, cancer, fragilex syndrome, sickle cell disease, lichen planus, vitiligo, chronicfatigue syndrome, etc. The pharmaceutical composition of the presentinvention may also be used as a thrombus-targeting/antithrombotic drugin treating thrombocytosis, myeloproliferative disease, polycythemiavera, budd-chiari syndrome, etc.

The pharmaceutical compositions/compounds of the present inventionsimultaneously have OH, NO and superoxide anionic free radicalscavenging, thrombolytic, as well as thrombus-targeting/antithromboticfunctions. Accordingly, they may remain therapeutically effective inpatients beyond 4 hours from the onset of stroke symptoms, i.e. theiruse is not limited by the 3-hour treatment window of tPA. Moreover, theuse of the pharmaceutical compositions/compounds of the presentinvention will not cause systemic bleeding reactions as tPA does, andmay scavenge the massive amount of free radicals of OH, NO, andsuperoxide anions during the ischemia-reperfusion process so as toprevent brain tissue damages for patients being treated. Since the twoC₁₋₄ alkyl groups and the linking arm are respectively introduced in thepositions 1 and 3 of the compounds having formula II, as compared with“the 6,7-dihydroxyisoquinoline having thrombolytic activity,” “the novelbinary conjugate of the present invention” shows better thrombolyticactivity, unique free radical scavenging and antithrombotic activity ata low dose, as well as excellent therapeutic effect in treating strokebeyond 4 hours from the stroke onset at a higher dose.

In the issued Chinese Patent, No. CN101497651B, “the6,7-dihydroxyisoquinoline having thrombolytic activity” was shown tohave thrombolytic activity at a dose of 10 nmol/kg. However, thecompounds of the present invention have good thrombolytic andantithrombotic activity at a dose of 0.1 nmol/kg. Moreover, thecompounds of the present invention have significant therapeutic effectin treating stroke beyond 4, 6, and 24 hours from the stroke onset at adose of 1, 2.5, and 5 μmol/kg, respectively.

In the preparation method of the compounds of the present invention, thepeptide Q having thrombolytic activity may be prepared first and thencoupled to the second group for linking of the linking arm T,alternatively, one or more of the amino acids of the peptide Q havingthrombolytic activity may be sequentially coupled to the linking arm Tin a predetermined order. For example, the first amino acid at oneterminal of the thrombolytic peptide Q is coupled to the second groupfor linking of the linking arm T, and one or more of the rest of theamino acids are then sequentially coupled thereto.

The preparation method of the present invention is described in moredetail below for further understanding.

The compound of formula II may be prepared by the following synthesisroute:

wherein R₁ and R₂ both are C₁₋₄ alkyl group and may be the same ordifferent. For example, 3,4-dihydroxy-L-phenylalanine (SM-1) and thecompound SM-2 are dissolved in TFA, and the3,4-dihydroxy-L-phenylalanine and SM-2 undergo Pictet-Spenglercondensation to obtain the compound having formula II in the presence ofanhydrous magnesium sulfate.

In a preferred embodiment, the linking arm in the preparation method ofthe invention is L-Lys, and the peptide having thrombolytic activity isa tripeptide having a sequence of PA (Pro-Ala). For example, thecarboxyl group of the compound II is coupled to a N-terminal of theL-Lys, and then the tripeptide containing the PA sequence is coupled tothe rest N-terminal or the C-terminal of the L-Lys linking arm. In someembodiments, when the R₁ and R₂ both are methyl group (i.e.3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-carboxylicacid), the linking arm is L-Lys, and the peptide having thrombolyticactivity is the tripeptide containing the PA (Pro-Ala) sequence, thecompound Ia, Ib, Ic, or Id may be obtained according to the preparationmethod of the present invention.

When the compound Ia is prepared according to the preparation method ofthe present invention, the synthesis route disclosed in FIG. 1 may bereferred.

In the embodiment shown in FIG. 1, the tripeptide containing the PAsequence is synthesized first and then coupled to the L-Lys linking arm,wherein the AA is selected from L-Ala, L-Val, L-Trp, L-Tyr, L-Pro,L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln, L-Asn, L-Asp, andL-Glu residue (respectively corresponding to compounds 5Aa-p,). Thereaction conditions are listed below: i) Acetone, TFA, MgSO₄; ii) HCl,Lys(Boc)-OBzl, DCC, HOBt, NMM; iii) 4M HCl/EA, ice bath; iv) DCC, HOBt,NMM, v) EtOH, Pd/C; 4M HCl/EA, ice bath; vi) EtOH, Pd/C; vii) 2M NaOH.In another embodiment, one amino acid of the tripeptide containing thePA sequence (such as AA) is coupled to the L-Lys linking arm, and therest two amino acids (such as Pro-Ala) of the tripeptide containing thePA sequence are then coupled to AA.

For example, in an embodiment of forming compound Ia, the preparationmethod of the present invention may include the following steps:

1) In the presence of TFA and anhydrous magnesium sulfate, making3,4-dihydroxy-L-phenylalanine and acetone undergo Pictet-Spenglercondensation to obtain3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-carboxylicacid;

2) In the presence of dicyclohexyl carbodiimide (DCC) andN-hydroxybenzotriazole triazole (HOBt), condensing the3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-carboxylicacid and HCl.Lys(Boc)-OBzl, in anhydrous N,N-dimethylformamide (DMF), toform3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys(Boc)-Obzl.In the condensation reaction, N-methylmorpholine (NMM) was used toconstantly adjust the mixture to pH=9;

3) Removing Boc from the3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys(Boc)-OBzlin a ethyl acetate solution of HCl to obtain3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys-OBzl;

4) In the presence of DCC and HOBt, condensing Boc-Pro withTos.Ala-Obzl, in anhydrous THF, to form Boc-Pro-Ala-OBzl;

5) In EtOH, hydrogenolyzing the Boc-Pro-Ala-OBzl to form Boc-Pro-Ala;

6) In the presence of DCC and HOBt, condensing the Boc-Pro-Ala withAA-Obzl, in anhydrous THF, to form Boc-Pro-Ala-AA-OBzl (AA is selectedfrom L-Ala, Gly, L-Phe, L-Val, L-Leu, L-Ile, L-Trp, L-Ser, L-Thr, L-Tyr,L-Lys(Z), L-Pro, L-Asn, and L-Gln residues);

7) In EtOH, hydrogenolyzing the Boc-Pro-Ala-AA-OBzl of step 6 to formBoc-Pro-Ala-AA;

8) In the presence of DCC and HOBt, condensing the3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys-OBzlwith the Boc-Pro-Ala-AA of step 6, in anhydrous DMF, to form3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys(Boc-Pro-Ala-AA)-OBzl(the definition of AA is the same as that in step 6);

9) In the presence of DCC and HOBt, condensing the3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys-OBzlwith Boc-AA(OBzl), in anhydrous DMF, to form3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys[Boc-AA(OBzl)]-OBzl(AA is selected from L-Asp, L-Glu residues);

10) In ethyl acetate solution of HCl, removing Boc from the3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys[Boc-AA(OBzl)]-OBzl to form 3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys[AA(OBzl)]-OBzl (the definition of AA is the sameas that in step 9);

11) In the presence of DCC and HOBt, condensing the3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys[AA(OBzl)]-OBzl with Boc-Pro-Ala, in anhydrous DMF, to form3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys[Boc-Pro-Ala-AA(OBzl)]-OBzl(the definition of AA is the same as that in step 9);

12) By hydrogenolysis and the removal of Boc, deprotecting the both3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys(Boc-Pro-Ala-AA)-OBzl(the definition of AA is the same as that in step 6) and the3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys[Boc-Pro-Ala-AA(OBzl)]-OBzl(the definition of AA is the same as that in step 9) to obtain3S-6,7-dihydroxy-1, 1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys(Pro-Ala-AA).

When compound Ib of the present invention is prepared according to thepreparation method of the present invention, synthesis route shown inFIG. 2 may be referred, wherein AA is selected from L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu residue (respectively corresponding to compounds5Ba-p). The reaction conditions are listed below: i) Acetone, TFA,MgSO4; ii) HCl, Lys(Boc)-OBzl, DCC, HOBt, NMM; iii) EtOH, Pd/C; iv) DCC,HOBt, NMM, v) EtOH, Pd/C; 4M HCl/EA, ice bath; vi) EtOH, Pd/C; vii) 4MHCl/EA, ice bath.

When compound Ic of the present invention is prepared according to thepreparation method of the present invention, synthesis route shown inFIG. 3 may be referred, wherein AA is selected from L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu residue (respectively corresponding to compounds5Ca-p). The reaction conditions are listed below: i) Acetone, TFA,MgSO₄; ii) HCl, Boc-Lys-OBzl, DCC, HOBt, NMM; iii) 4M HCl/EAice bath;iv) DCC, HOBt, NMM, v) EtOH, Pd/C; 4M HCl/EA, ice bath; vi) EtOH, Pd/C;vii) EtOH, Pd/C.

When compound Id of the present invention is prepared according to thepreparation method of the present invention, synthesis route shown inFIG. 4 may be referred, wherein AA is selected from L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu residue (respectively corresponding to compounds5 Da-p). The reaction conditions are listed below: i) Acetone, TFA,MgSO₄; ii) HCl, Boc-Lys-OBzl, DCC, HOBt, NMM; iii) EtOH, Pd/C; iv) DCC,HOBt, NMM, v) EtOH, Pd/C; 4M HCl/EA, ice bath; vi) EtOH, Pd/C; vii)EtOH, Pd/C.

In another preferred embodiment, the linking arm is L-Asp, and thepeptide having thrombolytic activity is a tripeptide containing a PA(Pro-Ala) sequence. For example, the carboxyl group of the compoundhaving formula II is coupled to the N-terminal of L-Asp, and then thetripeptide containing the PA sequence is coupled to one of the restC-terminal of the L-Asp linking arm. In some embodiments, when the R₁and R₂ of the compound having formula II both are methyl group (i.e.3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-carboxylicacid), the linking arm is L-Asp, and the peptide having thrombolyticactivity is the tripeptide containing the PA (Pro-Ala) sequence, thepreparation method of the present invention may form the compounds Ie orIf above.

When compound Ie of the present invention is prepared according to thepreparation method of the present invention, synthesis route shown inFIG. 5 may be referred, wherein AA is selected from L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu residue (respectively corresponding to compounds5Ea-p). The reaction conditions are listed below: i) Acetone, TFA,MgSO4; ii) HCl, Asp(OCH₃)-OBzl, DCC, HOBt, NMM; iii) EtOH, Pd/C; iv)DCC, HOBt, NMM, v) EtOH, Pd/C; 2M NaOH, ice bath; vi) EtOH, Pd/C; vii)4M HCl/EA, ice bath.

When compound If of the present invention is prepared according to thepreparation method of the present invention, synthesis route shown inFIG. 6 may be referred, wherein AA is selected from L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu residue (respectively corresponding to compounds5Fa-p). The reaction conditions are listed below: i) Acetone, TFA,MgSO4; ii) HCl, Asp(OBzl)-OCH₃, DCC, HOBt, NMM; iii) 2M NaOH, iv) DCC,HOBt, NMM, v) EtOH, Pd/C; 2M NaOH, ice bath; vi) EtOH, Pd/C; vii) 4MHCl/EA, ice bath.

In another preferred embodiment of the present invention, the linkingarm is L-Glu, and the peptide having thrombolytic activity is atripeptide containing a PA (Pro-Ala) sequence. For example, the carboxylgroup of the compound having formula II is coupled to the N-terminal ofL-Glu, and then the tripeptide containing the PA sequence is coupled toone of the rest C-terminal of the linking arm L-Glu. In someembodiments, when the R₁ and R₂ of the compound having formula II bothare methyl groups (i.e.3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-carboxylicacid), the linking arm is L-Glu, and the peptide having thrombolyticactivity is the tripeptide containing the PA (Pro-Ala) sequence, thepreparation method of the present invention may form the compounds Ig orIh above.

When compound Ig of the present invention is prepared according to thepreparation method of the present invention, synthesis route shown inFIG. 7 may be referred, wherein AA is selected from L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu residue (respectively corresponding to compounds5Ga-p). The reaction conditions are listed below: i) Acetone, TFA,MgSO4; ii) HCl, Glu(O CH₃)-OBzl, DCC, HOBt, NMM; iii) EtOH, Pd/C; iv)DCC, HOBt, NMM; v) EtOH, Pd/C; 2M NaOH, ice bath; vi) EtOH, Pd/C; vii)4M HCl/EA, ice bath.

When compound Ih of the present invention is prepared according to thepreparation method of the present invention, synthesis route shown inFIG. 8 may be referred, wherein AA is selected from L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu residue (respectively corresponding to compounds5Ha-p). The reaction conditions are listed below: i) Acetone, TFA,MgSO4; ii) HCl, Glu(O Bzl)-OCH₃, DCC, HOBt, NMM; iii) EtOH, Pd/C; iv)DCC, HOBt, NMM; v) EtOH, Pd/C; 2M NaOH, ice bath; vi) EtOH, Pd/C; vii)4M HCl/EA, ice bath.

The in vivo tests in rats of the compounds and pharmaceuticalcompostions of the present invention show that the compounds andpharmaceutical compostions of the present invention have excellentthrombolytic and antithombotic activity at low dose, and may effectivelyprotect the neurological functions of the stroke rats. Therefore, thecompounds and pharmaceutical compostions of the present invention mayeffectively and safely treat thrombotic diseases in clinical practice.

The present invention will now be described in connection with thefollowing specific examples, and the advantages and features thereofwill become apparent in view of the description. These examples aremerely illustrative and in no way limit the scope of the presentinvention. A person skilled in the art can understand that modificationor substitution may be made in details and formality of the technicalsolutions of the present invention without deviating from the spirit andscope of the present invention, and these modifications or substitutionsare intended to be within the scope of protection of the presentinvention.

Examples 1-68 illustrate the preparation methods in FIG. 1 for preparingcompounds 5Aa-p of the present invention.

Example 1: General Procedure for Synthesizing Peptide in Liquid Phase

An amino acid having a protected N-terminal was dissolved in anhydroustetrahydrofuran (THF), and the obtained solution was added withN-hydroxybenzotriazole triazole (HOBt). Ice bath the solution.N,N-dicyclohexyl carbodiimide (DCC) dissolved in anhydrous THF wasslowly added thereto, and then stirred at 0° C. for 15 minutes to obtainthe reaction solution (I). An amino acid having a protected C-terminalwas also dissolved in anhydrous THF while adjusted to pH 9 byN-methylmorpholine (NMM), and then mixed with the reaction solution (I)while maintained at pH9 by N-methylmorpholine (NMM). The reactionmixture was stirred at room temperature for 10 hours, and the reactionprogress was monitored by TLC. Until the starting material spotdisappeared as shown by TLC, the reaction mixture was filtered and thefiltrate was concentrated under reduced pressure. The obtained viscousconcentrate was dissolved by ethyl acetate (EA) or dichloromethane, theobtained solution was sequentially washed by 5% NaHCO₃ aqueous solution,5% KHSO₄ aqueous solution, and saturated NaCl aqueous solution. The EAor dichloromethane phase was dried by anhydrous magnesium sulfate,filtered, and the filtrate was concentrated under a reduced pressure toobtain the target compound.

Example 2: General Procedure for Removing Boc

Boc protected peptide was dissolved by a small amount of anhydrous ethylacetate, ethyl acetate solution of HCl (4 M) was added thereto andstirred on an ice bath until the starting material spot disappeared asshown by TLC. The reaction solution was repeatedly dried by a waterpump, and HCl gas was also removed completely. The residue wastriturated with petroleum ether or anhydrous ether repeatedly to obtainthe target compounds.

Example 3: General Procedure for Removing Benzyl Ester Group

Benzyl ester group protected polypeptide was dissolved by CH₃OH, NaOHaqueous solution (2M) was dropwise added thereto and stirred on an icebath, the reaction solution was maintained at 0° C. until the startingmaterial spot disappeared as shown by TLC. The reaction solution wasadjusted to neutral with 1 M HCl, and removed MeOH by reduced pressure.The reaction solution was acidified to pH 2 with 1 M HCl and thenextracted by EA. The combined EA phase was washed by saturated NaClaqueous solution to neutral, dried by anhydrous Na₂SO₄, and filtered.The filtrate was concentrated under reduced pressure to obtain thetarget compounds.

Example 4: General Procedure for Removing Carbobenzoxy Group or BenzylEster Group

Carbobenzoxy group or benzyl ester group protected peptide was dissolvedin suitable amount of EtOH, Pd/C (10% of reactants) was added thereto,and hydrogen was introduced to perform hydrogenolysis reaction at roomtemperature. After reaction, the reaction mixture was filtered and thenconcentrated under reduced pressure to obtain the target compound.

Example 5: Preparation of Boc-Pro

5.75 g (50 mmol) L-Pro was dissolved in 1 mL water. 25 mL NaOH aqueoussolution (2M) was dropwise added thereto and stirred slowly on an icebath to obtain solution (I). 13.08 g (60 mmol) (Boc)₂O was dissolved in25 mL dioxane to obtain solution (II). Solution (II) was dropwise addedto solution (I) and stirred on an ice bath, and then NaOH aqueoussolution (2M) was dropwise added to adjust pH 9, and the mixture wasstirred on an ice bath. After 30 minutes, pH value of the mixture wasmeasured, and NaOH aqueous solution (2M) was used to maintain pH valueat 9. Water pump was used to extract the generated gas. After stirringfor 48 hours, the TLC monitoring (CH₂Cl₂: MeOH 20:1) showed that thereaction was completed. The mixture was concentrated under a reducedpressure to remove dioxane. The residue was dissolved by 5 mL water, andthen adjust to pH 2 by saturated KHSO₄ aqueous solution. The aqueoussolution was extracted by EA for 3 times. Combined EA phase was washedby 5% KHSO₄ for 3 times, and then washed by saturated NaCl aqueoussolution to neutral. The separated EA layer was dried by addinganhydrous sodium sulfate and then filtered. The filtrate wasconcentrated under reduced pressure. The residue was recrystallized byEA-petroleum ether to obtain 10.2 g (95%) of the title compound as acolorless crystal. ESI-MS (m/e): 214 [M−H].

Example 6: Preparation of Boc-Pro-Ala-OBzl

By following the method of Example 1, 5.19 g (68%) of the title compoundas colorless powder was prepared from 4.3 g (20.0 mmol) Boc-Pro and 8.45g (24.0 mmol) Tos.Ala-OBzl. ESI-MS (m/e): 377 [M+H]⁺.

Example 7: Preparation of Boc-Pro-Ala

By following the method of Example 4, 3.59 g (91%) of the title compoundas colorless powder was prepared from 5.19 g (13.8 mmol)Boc-Pro-Ala-OBzl. ESI-MS (m/e): 285 [M−H]⁻.

Example 8: Preparation of Boc-Pro-Ala-Ala-OBzl

By following the method of Example 1, 3.04 g (68%) of the title compoundas colorless powder was prepared from 3.00 g (10.49 mmol)Boc-Pro-Ala-OBzl and 3.26 g (12.03 mmol) Tos.Ala-OBzl. ESI-MS (m/e): 448[M+H]⁺.

Example 9: Preparation of Boc-Pro-Ala-Ala

By following the method of Example 3, 3.21 g (90%) of the title compoundwas prepared from 4.47 g (10 mmol) Boc-Pro-Ala-Ala-OBzl. ESI-MS (m/e):356 [M−H]⁻.

Example 10: Preparation of Boc-Pro-Ala-Val-OBzl

By following the method of Example 1, 3.28 g (69%) of the title compoundas colorless powder was prepared from 3.00 g (10.49 mmol) Boc-Pro-Alaand 3.58 g (12.01 mmol) Tos.Val-OBzl. ESI-MS (m/e): 476 [M+H]⁺.

Example 11: Preparation of Boc-Pro-Ala-Val

By following the method of Example 3, 3.54 g (92%) of the title compoundwas prepared from 4.75 g (10 mmol) Boc-Pro-Ala-Val-OBzl. ESI-MS (m/e):384 [M−H]⁻.

Example 12: Preparation of Boc-Pro-Ala-Trp-OBzl

By following the method of Example 1, 3.65 g (65%) of the title compoundas colorless powder was prepared from 3.00 g (10.49 mmol) Boc-Pro-Alaand 3.97 g (12.01 mmol) HCl.Trp-OBzl. ESI-MS (m/e): 563 [M+H]⁺.

Example 13: Preparation of Boc-Pro-Ala-Trp

By following the method of Example 3, 4.21 g (89%) of the title compoundwas prepared from 5.62 g (10 mmol) Boc-Pro-Ala-Trp-OBzl. ESI-MS (m/e):471 [M−H]⁻.

Example 14: Preparation of Boc-Pro-Ala-Tyr-OBzl

By following the method of Example 1, 3.73 g (69%) of the title compoundas colorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and4.43 g (10 mmol) Tos.Tyr-OBzl. ESI-MS (m/e): 540 [M+H]⁺.

Example 15: Preparation of Boc-Pro-Ala-Tyr

By following the method of Example 3, 4.13 g (92%) of the title compoundwas prepared from 5.39 g (10 mmol) Boc-Pro-Ala-Tyr-OBzl. ESI-MS (m/e):448 [M−H]⁻.

Example 16: Preparation of Boc-Pro-Ala-Phe-OBzl

By following the method of Example 1, 3.82 g (66%) of the title compoundas colorless powder was prepared from 3.00 g (10.0 mmol) Boc-Pro-Ala and2.92 g (11.0 mmol) Tos.Phe-OBzl. ESI-MS (m/e): 524 [M+H]⁺.

Example 17: Preparation of Boc-Pro-Ala-Phe

By following the method of Example 3, 3.94 g (91%) of the title compoundwas prepared from 5.23 g (10 mmol) Boc-Pro-Ala-Phe-OBzl. ESI-MS (m/e):432 [M−H]⁻.

Example 18: Preparation of Boc-Pro-Ala-Gly-OBzl

By following the method of Example 1, 2.90 g (67%) of the title compoundas colorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and3.37 g (10 mmol) Tos.Gly-OBzl. ESI-MS (m/e): 434 [M+H]⁺.

Example 19: Preparation of Boc-Pro-Ala-Gly

By following the method of Example 3, 2.98 g (87%) title compound wasprepared from 4.33 g (10 mmol) Boc-Pro-Ala-Gly-OBzl. ESI-MS (m/e): 342[M−H]⁻.

Example 20: Preparation of Boc-Pro-Ala-Ser-OBzl

By following the method of Example 1, 2.92 g (63%) of the title compoundas colorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and3.67 g (10 mmol) Tos.Ser-OBzl. ESI-MS (m/e): 464 [M+H]⁺.

Example 21: Preparation of Boc-Pro-Ala-Ser

By following the method of Example 3, 3.28 g (88%) of the title compoundwas prepared from 4.63 g (10 mmol) Boc-Pro-Ala-Ser-OBzl. ESI-MS (m/e):372 [M−H]⁻.

Example 22: Preparation of Boc-Pro-Ala-Ile-OBzl

By following the method of Example 1, 3.33 g (65%) of the title compoundas colorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and3.93 g (10 mmol) Tos.Ile-OBzl. ESI-MS (m/e): 490 [M+H]⁺.

Example 23: Preparation of Boc-Pro-Ala-Ile

By following the method of Example 3, 3.67 g (91%) of the title compoundwas prepared from 4.89 g (10 mmol) Boc-Pro-Ala-Ile-OBzl. ESI-MS (m/e):398 [M−H]⁻.

Example 24: Preparation of Boc-Pro-Ala-Thr-OBzl

By following the method of Example 1, 3.39 g (71%) of the title compoundas colorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and3.81 g (10 mmol) Tos.Thr-OBzl. ESI-MS (m/e): 478 [M+H]⁺.

Example 25: Preparation of Boc-Pro-Ala-Thr

By following the method of Example 3, 3.56 g (91%) of the title compoundwas prepared from 4.77 g (10 mmol) Boc-Pro-Ala-Thr-OBzl. ESI-MS (m/e):386 [M−H]⁻.

Example 26: Preparation of Boc-Pro-Ala-Lys(Z)-OBzl

By following the method of Example 1, 4.15 g (65%) of the title compoundas colorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and4.06 g (10 mmol) HCl.Lys(Z)-OBzl. ESI-MS (m/e): 639 [M+H]⁺.

Example 27: Preparation of Boc-Pro-Ala-Lys(Z)

By following the method of Example 3, 4.71 g (86%) of the title compoundwas prepared from 6.39 g (10 mmol) Boc-Pro-Ala-Lys(Z)-OBzl. ESI-MS(m/e): 547[M−H]⁻.

Example 28: Preparation of Boc-Pro-Ala-Leu-OBzl

By following the method of Example 1, 3.37 g (69%) of the title compoundas colorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and3.93 g (10 mmol) Tos.Leu-OBzl. ESI-MS (m/e): 490 [M+H]⁺.

Example 29: Preparation of Boc-Pro-Ala-Leu

By following the method of Example 3, 3.67 g (91%) of the title compoundwas prepared from 4.89 g (10 mmol) Boc-Pro-Ala-Leu-OBzl. ESI-MS (m/e):398 [M−H]⁻.

Example 30: Preparation of Boc-Pro-Ala-Gln-OBzl

By following the method of Example 1, 3.23 g (63%) the title compound ascolorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and 2.73g (10 mmol) HCl.Gln-OBzl. ESI-MS (m/e): 505 [M+H]⁺.

Example 31: Preparation of Boc-Pro-Ala-Gln

By following the method of Example 3, 3.73 g (90%) of the title compoundwas prepared from 5.04 g (10 mmol) Boc-Pro-Ala-Gln-OBzl. ESI-MS (m/e):413 [M−H]⁻.

Example 32: Preparation of Boc-Pro-Ala-Asn-OBzl

By following the method of Example 1, 3.04 g (61%) of the title compoundas colorless powder was prepared from 3.15 g (11 mmol) Boc-Pro-Ala and2.59 g (10 mmol) HCl.Asn-OBzl. ESI-MS (m/e): 491 [M+H]⁺.

Example 33: Preparation of Boc-Pro-Ala-Asn

By following the method of Example 3, 3.67 g (92%) of the title compoundwas prepared from 4.90 g (10 mmol) Boc-Pro-Ala-Asn-OBzl. ESI-MS (m/e):399 [M−H]⁻.

Example 34: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-carboxylicacid (1)

5.0 g (25 mmol) 3,4-dihydroxy-L-phenylalanine was dissolved in 250 mLacetone, and then 6.0 g (30 mmol) anhydrous magnesium sulfate was addedinto the obtained solution. After 30 minutes, 25 mL TFA was addedthereto on an ice bath. The mixture was stirred at room temperature for96 hours until the starting material spot was disappeared as shown byTLC (CH₂Cl₂: MeOH 1:1). The reaction solution was filtered. The filtratewas concentrated under reduced pressure and the residue was dissolved inacetone and continued to be concentrated under reduced pressure for 3times. 200 mL anhydrous ether was added to the residue to precipitatemassive amount of colorless solid. After suction filtration, 5.8 g (95%)of the title compound was obtained as colorless solid. ESI-MS (m/e): 238[M+H]⁺; ¹HNMR (300 MHz, DMSO-d₆) δ/ppm=6.61 (s, 1H), 6.45 (s, 1H), 3.70(dd, J=3.9, 11.4 Hz, 1H), 2.76 (dd, J=11.7, 15.3 Hz, 1H), 2.62 (m, 1H),1.41 (s, 3H), 1.32 (s, 3H).

Example 35: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc)-OBzl (2)

By following the method of Example 1, 1.19 g (5.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-carboxylic acid wasdissolved in 10 mL of anhydrous DMF. 675 mg (5.00 mmol)N-hydroxybenzotriazole triazole (HOBt) was added to the obtainedsolution. After 10 minutes, a solution of 1.20 g (5.83 mmol)dicyclohexyl carbodiimide (DCC) and 5 mL anhydrous DMF was added theretoon an ice bath to obtain reaction solution (I). 2.83 g (5.53 mmol)HCl.Lys(Boc)-OBzl was dissolved in 15 mL anhydrous DMF and then stirredfor 30 minutes to obtain reaction solution (II). The reaction solution(II) was added into the reaction solution (I) while stirred on an icebath. The reaction mix was stirred at room temperature for 12 hours, andadjusted to pH=9 using MMM when necessary until3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-carboxylicacid disappeared as shown by TLC (CH₂Cl₂: MeOH 10:1). The reactionmixture was filtered to remove Dicyclohexylurea (DCU). The filtrate wasconcentrated under reduced pressure to remove DMF. The residue wasdissolved in 150 mL EA. The obtained solution was sequentially washed bysaturated NaHCO₃ aqueous solution for 3 times, and then by saturatedNaCl aqueous solution for 3 times. The EA solution was dried byanhydrous Na₂SO₄, and filtered. The filtrate was concentrated to dryunder reduced pressure. The residue was purified by columnchromatography (CH₂Cl₂: MeOH, 50:1) to obtain 327 mg (59%) of the titlecompound as a light pink powder. ESI-MS (m/e): 556 [M+H]⁺; ¹HNMR (300MHz, DMSO-d₆) δ/ppm=8.64 (s, 1H), 8.52 (s, 1H), 8.14 (d, J=7.5 Hz, 1H),7.36 (m, 5H), 6.74 (m, 1H), 6.57 (s, 1H), 6.37 (s, 1H), 5.14 (m, 2H),4.30 (m, 1H), 3.55 (m, 2H), 3.32 (m, 2H), 2.88 (m, 2H), 2.57 (d, J=3.9Hz, 1H), 2.27 (m, 1H), 2.15 (s, 1H), 1.69 (m, 4H), 1.36 (s, 9H), 1.33(s, 3H), 1.25 (s, 3H).

Example 36: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzl(3A)

By following method of Example 2, 1.01 g (82%) of the title compound aslight pink solid was prepared from 1.50 g (2.73 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc)-Obzl, whichwas directly used in the next reaction. ESI-MS(m/e): 456[M+H]⁺.

Example 37: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Ala)-OBzl (4Aa)

By following the method of Example 1, 302 mg (38%) of the title compoundas light yellow powder was prepared from 482 mg (1.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 424 mg (1.20 mmol) Boc-Pro-Ala-Ala. ESI-MS (m/e): 795 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.15 (s, 1H), 8.89 (s, 2H), 8.11 (m, 1H), 7.94(m, 1H), 7.85 (m, 1H), 7.76 (m, 1H), 7.44-7.31 (m, 5H), 6.66 (s, 1H),6.47 (s, 1H), 5.14 (m, 2H), 4.25 (m, 1H), 4.23 (m, 3H), 4.12 (m, 1H),3.66 (m, 1H), 3.12 (m, 2H), 2.96 (m, 2H), 2.89-2.91 (m, 2H), 2.11-2.08(m, 1H), 1.77 (m, 5H), 1.59 (s, 3H), 1.47-1.27 (m, 17H), 1.27-1.08 (m,7H).

Example 38: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Val)-OBzl (4Ab)

By following the method of Example 1, 604 mg (37%) of the title compoundas light yellow powder was prepared from 964 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 924 mg (2.40 mmol) Boc-Pro-Ala-Val. ESI-MS (m/e): 823 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.18 (s, 1H), 8.93 (m, 2H), 8.05 (m, 2H), 7.42(m, 5H), 6.66 (s, 1H), 6.47 (s, 1H), 5.16 (s, 1H), 4.37-4.28 (m, 3H),4.11 (m, 3H), 3.06-2.89 (m, 5H), 2.75 (m, 6H), 2.04 (s, 1H), 1.91 (m,1H), 1.79 (m, 5H), 1.62 (s, 3H), 1.49 (s, 3H), 1.39 (m, 7H), 1.32 (m,3H), 1.21 (m, 4H), 0.82 (m, 6H).

Example 39: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Trp)-OBzl (4Ac)

By following the method of Example 1, 318 mg (35%) of the title compoundas light yellow powder was prepared from 482 mg (1.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 567 mg (1.20 mmol) Boc-Pro-Ala-Trp. ESI-MS (m/e): 910 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.51 (s, 1H), 9.26 (m, 2H), 8.98 (m, 2H), 8.05(d, 1H, J=7.2 Hz), 7.94 (m, 1H), 7.78 (d, 1H, J=7.2 Hz), 7.54 (m, 1H),7.38 (m, 5H), 7.31 (d, 1H, J=4.5 Hz), 7.24 (m, 1H), 7.11 (t, 1H, J=4.5Hz), 7.05 (t, 1H, J=4.5 Hz), 6.67 (s, 1H), 6.52 (s, 1H), 5.15 (s, 2H),4.44 (m, 1H), 4.35 (m, 2H), 4.07 (m, 2H), 3.23 (m, 1H), 3.17 (m, 5H),2.86 (m, 1H), 1.97 (m, 2H), 1.76-1.70 (m, 8H), 1.31 (s, 3H), 1.24 (s,3H), 1.93-1.79 (m, 9H), 1.16 (m, 4H).

Example 40: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Tyr)-OBzl (4Ad)

By following the method of Example 1, 549 mg (31%) of the title compoundas light yellow powder was prepared from 964 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 1077 mg (2.40 mmol) Boc-Pro-Ala-Tyr. ESI-MS (m/e): 887 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.17 (m, 1H), 9.02 (m, 1H), 8.73 (m, 1H), 8.51(m, 1H), 8.09 (m, 1H), 7.97 (m, 1H), 7.73 (m, 1H), 7.40 (m, 5H), 6.96(m, 2H), 6.61 (m, 3H), 6.48 (s, 1H), 5.16 (s, 2H), 4.23 (m, 2H), 4.07(m, 2H), 3.88 (m, 1H), 3.01 (m, 3H), 2.74 (m, 3H), 2.02 (m, 2H), 1.74(m, 5H), 1.46 (s, 3H), 1.36-1.24 (m, 16H), 1.16 (m, 3H), 0.87 (m, 1H).

Example 41: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Pro)-OBzl (4Ae)

By following the method of Example 1, 607 mg (37%) of the title compoundas light yellow powder was prepared from 964 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 957 mg (2.40 mmol) Boc-Pro-Ala-Pro. ESI-MS (m/e): 821 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.54 (m, 1H), 9.23 (s, 1H), 9.11 (m, 1H), 8.96(s, 1H), 8.05 (m, 1H), 7.85 (m, 1H), 7.40 (m, 5H), 6.67 (s, 1H), 6.48(s, 1H), 5.16 (m, 2H), 4.54 (m, 1H), 4.38 (m, 2H), 4.24 (m, 1H), 4.10(m, 2H), 3.55 (m, 1H), 3.17 (m, 3H), 3.05 (m, 3H), 2.94 (m, 1H), 1.96(m, 2H), 1.79 (m, 7H), 1.64 (s, 3H), 1.51 (s, 3H), 1.38-1.32 (m, 13H),1.20 (m, 4H).

Example 42: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Phe)-OBzl (4Af)

By following the method of Example 1, 626 mg (36%) of the title compoundas light yellow powder was prepared from 964 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 1039 mg (2.40 mmol) Boc-Pro-Ala-Phe. ESI-MS (m/e): 871 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.24 (s, 1H), 9.09 (m, 2H), 8.96 (s, 1H),8.01-7.93 (m, 3H), 7.38 (m, 5H), 7.21 (m, 5H), 6.67 (s, 1H), 6.49 (s,1H), 5.17 (s, 2H), 4.38 (m, 3H), 4.09 (m, 2H), 3.28 (m, 4H), 2.88 (m,2H), 2.79 (m, 5H), 1.99 (m, 2H), 1.74 (m, 8H), 1.50 (m, 3H), 1.32 (m,14H), 1.16 (m, 4H).

Example 43: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Gly)-OBzl(4Ag)

By following the method of Example 1, 304 mg (39%) of the title compoundas light yellow powder was prepared from 482 mg (1.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 412 mg (1.20 mmol) Boc-Pro-Ala-Gly. ESI-MS (m/e): 781 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.39 (s, 1H), 8.97 (m, 2H), 8.24-8.14 (m, 2H),7.79 (m, 1H), 7.41 (m, 5H), 6.66 (s, 1H), 6.47 (s, 1H), 5.16 (s, 2H),4.35 (m, 2H), 4.21 (m, 2H), 3.71-3.66 (m, 2H), 3.03 (m, 3H), 2.89-2.76(m, 1H), 2.07 (m, 1H), 1.81-1.77 (m, 6H), 1.62 (s, 3H), 1.50 (s, 3H),1.38 (m, 7H), 1.32 (m, 8H), 1.23 (m, 4H).

Example 44: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Ser)-OBzl(4Ah)

By following the method of Example 1, 454 mg (29%) of the title compoundas light yellow powder was prepared from 965 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 896 mg (2.40 mmol) Boc-Pro-Ala-Ser. ESI-MS (m/e): 811 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.48 (m, 1H), 9.23 (s, 2H), 8.96 (m, 1H), 8.19(m, 1H), 7.40 (m, 5H), 6.67 (s, 1H), 6.48 (s, 1H), 5.16 (m, 2H), 4.92(m, 1H), 4.31 (m, 3H), 4.13 (m, 3H), 3.53 (m, 2H), 3.16 (m, 3H),3.04-2.81 (m, 4H), 2.08 (m, 1H), 1.78 (m, 5H), 1.63 (s, 3H), 1.51 (s,3H), 1.39-1.32 (m, 14H), 1.22 (m, 3H).

Example 45: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Ile)-OBzl(4Ai)

By following the method of Example 1, 310 mg (37%) of the title compoundas light yellow powder was prepared from 482 mg (1.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 479 mg (1.20 mmol) Boc-Pro-Ala-Ile. ESI-MS (m/e): 837 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=8.84-8.68 (m, 2H), 8.37 (s, 1H), 7.99-7.87 (m,3H), 7.39 (m, 4H), 6.60 (s, 1H), 6.39 (s, 1H), 5.14 (m, 2H), 4.32 (m,2H), 4.17 (m, 2H), 3.65 (m, 1H), 3.54 (m, 1H), 3.17 (m, 1H), 2.96 (m,2H), 2.68 (m, 3H), 2.11 (m, 1H), 1.74-1.68 (m, 7H), 1.31-1.21 (m, 11H),1.17 (m, 5H), 1.08 (m, 1H), 0.82 (m, 8H).

Example 46: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Thr)-OBzl(4Aj)

By following the method of Example 1, 495 mg (30%) of the title compoundas light yellow powder was prepared from 964 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 929 mg (2.40 mmol) Boc-Pro-Ala-Thr. ESI-MS (m/e): 825 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.18 (s, 1H), 8.91 (s, 2H), 8.27 (m, 1H), 7.64(m, 1H), 7.41 (m, 6H), 6.65 (s, 1H), 6.48 (s, 1H), 5.16 (s, 2H), 4.35(m, 3H), 4.23 (m, 3H), 3.07 (m, 3H), 2.72 (m, 1H), 1.83-1.76 (m, 5H),1.61 (s, 3H), 1.48 (s, 3H), 1.38-1.32 (m, 14H), 1.23 (m, 3H), 1.11 (m,3H).

Example 47: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys[Boc-Pro-Ala-Lys(Z)]-OBzl(4Ak)

By following the method of Example 1, 750 mg (30%) of the title compoundas light yellow powder was prepared from 964 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 1209 mg (2.40 mmol) Boc-Pro-Ala-Lys(Z). ESI-MS (m/e): 987 [M+H]⁺;¹HNMR (300 MHz, DMSO-d₆) δ/ppm=8.68 (s, 1H), 8.56 (s, 1H), 8.16 (m, 1H),8.04 (m, 1H), 7.78 (m, 1H), 7.68 (m, 1H), 7.34 (m, 10H), 6.58 (s, 1H),6.37 (s, 1H), 5.14 (s, 2H), 4.98 (s, 2H), 4.29 (m, 2H), 4.09 (m, 3H),3.56 (m, 3H), 3.01 (m, 4H), 2.62 (m, 1H), 2.40 (m, 1H), 2.27 (m, 1H),2.15 (s, 1H), 2.06 (m, 1H), 2.01 (s, 1H), 1.78 (m, 6H), 1.38-1.31 (m,18H), 1.17 (m, 10H).

Example 48: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Leu)-OBzl(4Al)

By following the method of Example 1, 301 mg (36%) of the title compoundas light yellow powder was prepared from 482 mg (1.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 479 mg (1.20 mmol) Boc-Pro-Ala-Leu. ESI-MS (m/e): 837 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=8.69-8.57 (m, 2H), 8.18-8.06 (m, 2H), 7.85 (m,1H), 7.69 (m, 5H), 6.58 (s, 1H), 6.37 (s, 1H), 5.14 (m, 2H), 4.25-4.11(m, 5H), 3.63-3.55 (m, 1H), 3.17 (s, 2H), 3.04-2.98 (m, 2H), 2.64-2.59(m, 1H), 2.15-2.07 (m, 1H), 1.78-1.67 (m, 5H), 1.36 (m, 14H), 1.28 (m,4H), 1.19 (m, 6H).

Example 49: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Gln)-OBzl(4Am)

By following the method of Example 1, 665 mg (39%) of the title compoundas light yellow powder was prepared from 965 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 994 mg (2.40 mmol) Boc-Pro-Ala-Gln. ESI-MS (m/e): 852 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.3-9.1 (m, 4H), 8.16 (m, 1H), 7.96 (m, 2H),7.83 (m, 1H), 7.44-7.38 (m, 7H), 6.89 (s, 1H), 6.86 (s, 1H), 6.56 (s,1H), 5.17 (s, 2H), 4.4-4.1 (m, 6H), 3.17 (m, 4H), 2.98-2.83 (m, 2H),2.08 (m, 4H), 1.91 (m, 1H), 1.64 (s, 3H), 1.51 (s, 3H), 1.38-1.31 (m,17H), 1.22-1.20 (m, 4H).

Example 50: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Asn)-OBzl(4An)

By following the method of Example 1, 636 mg (38%) of the title compoundas light yellow powder was prepared from 965 mg (2.01 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzland 960 mg (2.40 mmol) Boc-Pro-Ala-Asn. ESI-MS (m/e): 838 [M+H]⁺; ¹HNMR(300 MHz, DMSO-d₆) δ/ppm=9.26 (s, 1H), 9.04 (m, 2H), 8.31 (m, 1H), 8.03(m, 1H), 7.57 (m, 1H), 7.44 (m, 6H), 6.90 (m, 1H), 6.68 (s, 1H), 6.49(s, 1H), 5.16 (s, 2H), 4.45-4.40 (m, 3H), 4.14 (m, 2H), 3.42 (m, 5H),3.17 (m, 3H), 2.89-2.82 (m, 1H), 2.09 (m, 1H), 1.90 (m, 1H), 1.79-1.74(m, 6H), 1.64 (s, 3H), 1.52 (s, 3H), 1.39-1.32 (m, 15H), 1.22 (m, 3H).

Example 51: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys[Boc-Pro-Ala-Asp(OBzl)]-OBzl(4Ao)

By following the method of Example 1, 403 mg (35%) of the title compoundas light yellow powder was prepared from 870 mg (1.24 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys[Asp(OBzl)]-OBzland 429 mg (2.48 mmol) Boc-Pro-Ala. ESI-MS (m/e): 929 [M+H]⁺; ¹HNMR (300MHz, DMSO-d₆) δ/ppm=8.32 (m, 1H), 8.10 (m, 1H), 7.96 (m, 1H), 7.36 (m,10H), 6.60 (s, 1H), 6.39 (s, 1H), 5.14 (s, 2H), 5.06 (s, 2H), 4.57 (m,1H), 4.32 (m, 1H), 4.23 (m, 1H), 4.11 (m, 2H), 3.74 (m, 1H), 3.55 (m,1H), 3.34-3.28 (m, 2H), 3.00 (m, 2H), 2.89 (s, 1H), 2.81 (m, 1H), 2.73(m, 2H), 2.58 (m, 1H), 2.16-2.06 (m, 1H), 1.91 (s, 2H), 1.75 (m, 5H),1.40-1.32 (m, 22H), 1.17 (m, 3H).

Example 52: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys[Boc-Pro-Ala-Glu(OBzl)]-OBzl(4Ap)

By following the method of Example 1, 269 mg (38%) of the title compoundas light yellow powder was prepared from 530 mg (0.75 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys[Glu(OBzl)]-OBzl and 256 mg (0.90 mmol) Boc-Pro-Ala. ESI-MS (m/e): 943[M+H]⁺; ¹HNMR (300 MHz, DMSO-d₆) δ/ppm=9.26 (s, 1H), 9.10 (m, 1H), 8.97(s, 1H), 8.79-7.88 (m, 3H), 7.36 (m, 10H), 6.67 (s, 1H), 6.48 (s, 1H),5.14 (s, 2H), 5.06 (s, 2H), 4.35 (m, 1H), 4.23 (m, 2H), 4.10 (m, 1H),3.83 (s, 1H), 3.34 (m, 5H), 2.83 (m, 1H), 2.72 (m, 1H), 2.34 (m, 2H),1.78 (m, 10H), 1.51 (s, 3H), 1.36-1.31 (m, 14H), 1.21 (m, 3H).

Example 53: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Ala)(5Aa)

First, the benzyl ester group was removed from 300 mg (0.38 mmol) of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Ala)-OBzlby following the method of Example 4. Next, Boc was removed by followingthe method of Example 2 to give 266 mg (78%) of the title compound ascolorless powder. p 207.2-209.4° C.; [α]_(D) ²⁵=−4.5 (c=0.25, CH₃OH);ESI-MS (m/e): 630 [M−H]⁻; IR(KBr): 3221.1, 3057.2, 2983.8, 2943.3,2362.8, 1660.7, 1546.9, 1533.4, 1448.5, 1379.1, 1240.2, 1049.3, 867.9,659.7; ¹HNMR (300 MHz, D₂O) δ/ppm=6.79 (s, 1H), 6.68 (s, 1H), 4.36-4.29(m, 5H), 3.32-3.12 (m, 6H), 1.89 (m, 4H), 1.70 (s, 3H), 1.57 (s, 3H),1.48 (m, 2H), 1.33 (m, 9H).

Example 54: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Val)(5Ab)

By following the method of Example 53, 187 mg (81%) of the titlecompound as colorless powder was prepared from 300 mg (0.36 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Val)-OBzl. Mp 195.8-197.9° C.; [α]_(D) ²⁵=−3.5 (c=0.25, CH₃OH); ESI-MS(m/e): 630 [M−H]⁻; IR(KBr): 3221.1, 3062.9, 2970.4, 1658.8, 1546.9,1533.4, 1450.5, 1384.9, 1240.2, 1047.4, 991.41, 866.04, 671.2; ¹HNMR(300 MHz, D₂O) δ/ppm=8.13 (m, 1H), 6.79 (s, 1H), 6.68 (s, 1H), 4.36-4.29(m, 4H), 3.89 (m, 1H), 3.36 (m, 2H), 3.23 (m, 2H), 3.08 (m, 2H), 1.89(m, 4H), 1.70 (s, 3H), 1.57 (s, 3H), 1.48 (m, 1H), 1.37 (m, 6H), 1.32(m, 2H), 0.89 (m, 6H).

Example 55: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Trp)(5Ac)

By following the method of Example 53, 180 mg (76%) of the titlecompound as light pink powder was prepared from 300 mg (0.33 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Trp)-OBzl. Mp 205.1-207.9° C.; [α]_(D) ²⁵=−1.3 (c=0.25, CH₃OH); ESI-MS(m/e): 718 [M−H]⁻; IR(KBr): 3221.2, 3059.1, 2981.9, 2941.4, 1660.7,1533.4, 1448.5, 1388.7, 1240.2, 867.9, 748.4, 630.7; ¹HNMR (300 MHz,D₂O) δ/ppm=7.50 (d, J=7.8 Hz, 1H), 7.39 (d, J=7.8 Hz, 1H), 7.12 (t,J=7.2 Hz, 1H), 7.05 (t, J=7.2 Hz, 1H), 6.74 (s, 1H), 6.62 (s, 1H), 4.38(m, 1H), 4.35 (m, 1H), 4.27 (m, 1H), 4.18 (m, 2H), 3.24 (m, 2H), 3.16(m, 3H), 3.06 (m, 3H), 1.83 (m, 1H), 1.68 (m, 4H), 1.47 (m, 3H), 1.37(m, 2H), 1.25 (m, 3H), 1.21 (m, 3H).

Example 56: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Tyr)(5Ad)

By following the method of Example 53, 174 mg (74%) of the titlecompound as light yellow powder was prepared from 303 mg (0.34 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Tyr)-OBzl. Mp 200.5-202.8° C.; [α]_(D) ²⁵=−2.3 (c=0.25, CH₃OH); ESI-MS(m/e): 695 [M−H]⁻; IR(KBr): 3215.3, 3055.2, 2983.8, 2947.2, 1658.8,1548.8, 1523.8, 1448.5, 1384.9, 1238.3, 1163.1, 657.7; ¹HNMR (300 MHz,D₂O) δ/ppm=6.98 (m, 2H), 6.92 (m, 3H), 6.65 (s, 1H), 4.34-4.26 (m, 6H),3.27 (m, 2H), 3.15 (m, 1H), 3.08 (m, 1H), 2.96 (m, 2H), 2.85 (m, 12H),1.89 (m, 5H), 1.70 (s, 3H), 1.51 (m, 3H), 1.33 (m, 3H), 1.25 (m, 4H).

Example 57: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Pro)(5Ae)

By following the method of Example 53, 179 mg (78%) of the titlecompound as light yellow powder was prepared from 301 mg (0.36 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Pro)-OBzl.Mp 204.8-205.4° C.; [α]_(D) ²⁵=−5.0 (c=0.25, CH₃OH); ESI-MS (m/e): 629[M−H]⁻; IR(KBr): 3244.3, 3070.7, 2980.1, 2947.2, 1662.6, 1639.5, 1554.6,1450.5, 1379.1, 1246.1, 1201.6, 1047.3, 995.3, 871.8, 657.7; ¹HNMR (300MHz, D₂O) δ/ppm=6.78 (s, 1H), 6.68 (s, 1H), 4.57 (m, 1H), 4.39-4.33 (m,5H), 3.74 (m, 1H), 3.60 (m, 1H), 3.26 (m, 2H), 3.17 (m, 1H), 3.04 (m,2H), 2.19 (m, 3H), 1.85 (m, 5H), 1.69 (m, 4H), 1.54 (m, 6H), 1.36 (m,6H).

Example 58: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Phe)(5Af)

By following the method of Example 53, 183 mg (78%) of the titlecompound as light yellow powder was prepared from 300 mg (0.34 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Phe)-OBzl.Mp 175.8-178.2° C.; [α]_(D) ²⁵=−3.3 (c=0.25, CH₃OH); ESI-MS (m/e): 679[M−H]⁻; IR(KBr): 3215.3, 3049.5, 2949.2, 1662.6, 1539.2, 1454.3, 1394.5;¹HNMR (300 MHz, D₂O) δ/ppm=7.26 (m, 3H), 7.14 (m, 2H), 6.77 (s, 1H),6.65 (s, 1H), 4.39-4.33 (m, 3H), 4.03 (m, 3H), 3.42 (m, 1H), 3.37 (m,1H), 3.29 (m, 2H), 3.17 (m, 1H), 3.09 (m, 4H), 2.95 (m, 3H), 1.91 (m,5H), 1.74 (s, 3H), 1.53 (s, 3H), 1.33 (m, 6H).

Example 59: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Gly)(5Ag)

By following the method of Example 53, 193 mg (85%) of the titlecompound as colorless powder was prepared from 300 mg (0.38 mmol)S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Gly)-OBzl.Mp 196.4-199.5° C.; [α]_(D) ²⁵=−3.0 (c=0.25, CH₃OH); ESI-MS (m/e): 588[M−H]⁻; IR(KBr): 3223.1, 3062.9, 2983.8, 1630.2, 1552.7, 1537.3, 1448.5,1382.9, 1244.1, 1049.3, 1006.8, 869.9, 661.6; ¹HNMR (300 MHz, D₂O)δ/ppm=6.77 (s, 1H), 6.67 (s, 1H), 4.34-4.28 (m, 4H), 3.78 (s, 2H), 3.30(m, 2H), 3.15 (m, 3H), 1.92 (m, 3H), 1.70 (s, 3H), 1.55 (s, 3H), 1.33(m, 6H).

Example 60: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Ser)(5Ah)

By following the method of Example 53, 181 mg (79%) of the titlecompound as light yellow powder was prepared from 300 mg (0.37 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Ser)-OBzl.Mp 195.6-197.4° C.; [α]_(D) ²⁵=−3.8 (c=0.25, CH₃OH); ESI-MS (m/e): 619[M−H]⁻; IR(KBr): 3228.8, 3061.0, 2981.9, 2943.4, 1732.1, 1662.6, 1550.71533.3, 1448.5, 1384.9, 1244.1, 1159.2, 1049.3, 869.9, 657.7, 518.8;¹HNMR (300 MHz, D₂O) δ/ppm=6.78 (s, 1H), 6.67 (s, 1H), 4.41-4.26 (m,5H), 3.74 (m, 2H), 3.25-3.13 (m, 6H), 1.91 (m, 3H), 1.69 (s, 3H), 1.55(m, 3H), 1.46 (m, 2H), 1.37 (m, 6H).

Example 61: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Ile)(5Ai)

By following the method of Example 53, 181 mg (78%) of the titlecompound as colorless powder was prepared from 302 mg (0.36 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Ile)-OBzl.Mp 175.6-178.4° C.; [α]_(D) ²⁵=−3.3 (c=0.25, CH₃OH); ESI-MS (m/e): 645[M−H]⁻; IR(KBr): 3224.9, 3057.2, 2970.4, 2758.2, 2497.8, 2360.87,1656.9, 1535.3, 1454.3, 1388.7, 1244.1, 1159.1, 1037.7, 871.8, 659.6;¹HNMR (300 MHz, D₂O) δ/ppm=6.79 (s, 1H), 6.68 (s, 1H), 4.42 (m, 1H),4.35 (m, 1H), 4.29 (m, 1H), 4.18 (m, 1H), 3.92 (d, J=1.2 Hz, 1H), 3.31(m, 2H), 3.19 (m, 2H), 3.10 (m, 3H), 2.34 (m, 1H), 2.12 (m, 1H), 1.74(m, 4H), 1.57 (m, 3H), 1.36 (m, 6H), 1.21 (m, 2H), 0.84 (m, 9H).

Example 62: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Thr)(5Aj)

By following the method of Example 53, 175 mg (76%) of the titlecompound as light yellow powder was prepared from 300 mg (0.36 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Thr)-OBzl. Mp 194.8-197.5° C.; [α]_(D) ²⁵=−2.8 (c=0.25, CH₃OH); ESI-MS(m/e): 633 [M−H]⁻; IR(KBr): 3236.5, 3066.8, 2933.7, 1660.7, 1548.8,1533.4, 1450.8, 1381.0, 1240.2, 1157.3, 1049.3, 869.9, 669.3; ¹HNMR (300MHz, D₂O) δ/ppm=6.81 (s, 1H), 6.71 (s, 1H), 4.43-4.31 (m, 4H), 4.11 (m,2H), 3.33 (m, 2H), 3.24 (m, 1H), 3.24 (m, 2H), 3.21 (m, 1H), 3.18 (m,1H), 3.09 (m, 1H), 1.94 (m, 3H), 1.74 (s, 3H), 1.58 (s, 3H), 1.51 (m,2H), 1.23 (m, 1H), 1.19 (m, 1H), 1.14 (m, 3H).

Example 63: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Lys)(5Ak)

By following the method of Example 53, 152 mg (75%) of the titlecompound as dark yellow powder was prepared from 305 mg (0.31 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys[Boc-Pro-Ala-Lysl(Z)]-OBzl.Mp 185.6-188.1° C.; [α]_(D) ²⁵=−1.8 (c=0.25, CH₃OH); ESI-MS (m/e): 660[M−H]⁻; IR(KBr): 3853.7, 3738.1, 2956.8, 2349.3, 2017.5, 1668.4, 1537.3,1400.3, 1257.6, 1043.5, 871.8, 671.2; ¹HNMR (300 MHz, D₂O) δ/ppm=6.80(s, 1H), 6.69 (s, 1H), 4.45 (m, 1H), 4.35 (m, 1H), 4.29 (m, 1H), 4.18(m, 2H), 3.73 (m, 2H), 3.22-3.13 (m, 8H), 3.09 (m, 4H), 2.95 (m, 1H),1.93 (m, 6H), 1.74 (m, 9H), 1.58 (m, 6H), 1.47 (m, 2H), 1.32-1.27 (m,16H), 1.18 (m, 2H).

Example 64: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Leu)(5Al)

By following the method of Example 53, 178 mg (77%) of the titlecompound as colorless powder was prepared from 300 mg (0.36 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Leu)-OBzl.Mp 189.7-192.7° C.; [α]_(D) ²⁵=−3.5 (c=0.25, CH₃OH); ESI-MS (m/e): 645[M−H]⁻; IR(KBr): 3224.9, 3064.9, 2956.8, 1660.7, 1548.8, 1535.3, 1448.5,1381.0, 1242.2, 1049.3, 997.2, 869.9, 669.3; ¹HNMR (300 MHz, D₂O)δ/ppm=6.78 (s, 1H), 6.68 (s, 1H), 4.38-4.29 (m, 3H), 4.13 (m, 2H), 3.31(m, 3H), 3.21 (m, 1H), 3.08 (m, 2H), 1.89 (m, 4H), 1.70 (s, 3H), 1.57(m, 4H), 1.46 (m, 5H), 1.32 (m, 6H), 1.32 (m, 1H), 1.16 (m, 6H).

Example 65: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Gln)(5Am)

By following the method of Example 53, 184 mg (79%) of the titlecompound as colorless powder was prepared from 300 mg (0.35 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Gln)-OBzl.Mp 181.3-183.3° C.; [α]_(D) ²⁵=−3.0 (c=0.25, CH₃OH); ESI-MS (m/e): 660[M−H]⁻; IR(KBr): 3213.4, 3059.1, 2981.9, 2943.4, 2362.8, 1739.8, 1662.6,1548.8, 1537.3, 1450.5, 1375.3, 1244.1, 1047.2, 871.8, 659.7; ¹HNMR (300MHz, D₂O) δ/ppm=6.80 (s, 1H), 6.69 (s, 1H), 4.40-4.29 (m, 5H), 3.33 (m,3H), 3.17 (m, 3H), 1.90 (m, 2H), 1.69 (s, 3H), 1.55 (s, 3H), 1.43 (m,2H), 1.31 (m, 8H).

Example 66: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Asn)(5An)

By following the method of Example 53, of 174 mg (78%) of the titlecompound as colorless powder was prepared from 302 mg (0.36 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Boc-Pro-Ala-Asn)-OBzl.Mp 202.5-204.7° C.; [α]_(D) ²⁵=−2.3 (c=0.25, CH₃OH); ESI-MS (m/e): 646[M−H]⁻; IR(KBr): 3207.6, 3059.1, 2943.3, 1674.2, 1537.3, 1448.5, 1381.0,1247.9, 642.3; ¹HNMR (300 MHz, D₂O) δ/ppm=6.78 (s, 1H), 6.67 (s, 1H),4.48 (m, 1H), 4.42-4.29 (m, 4H), 3.29 (m, 2H), 3.16 (m, 4H), 2.65 (m,2H), 1.90 (m, 2H), 1.69 (s, 3H), 1.55 (s, 3H), 1.31 (m, 6H).

Example 67: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Asp)(5Ao)

By following the method of Example 53, of 174 mg (83%) of the titlecompound as colorless powder was prepared from 300 mg (0.32 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-[Boc-Pro-Ala-Asp(OBzl)]-OBzl.Mp 207.1-209.7° C.; [α]_(D) ²⁵=−3.5 (c=0.25, CH₃OH); ESI-MS (m/e): 647[M−H]⁻; IR(KBr): 3383.3, 3061.0, 2360.9, 1670.3, 1550.8, 1448.5, 1400.3,1246.3, 873.8, 611.4; ¹HNMR (300 MHz, D₂O) δ/ppm=6.76 (s, 1H), 6.66 (s,1H), 4.52 (m, 1H), 4.37-4.281 (m, 4H), 3.27 (m, 4H), 3.13 (m, 1H), 3.05(m, 1H), 2.76 (m, 2H), 1.97 (m, 1H), 1.90 (m, 1H), 1.71 (s, 3H), 1.55(s, 3H), 1.43 (m, 2H), 1.31 (m, 6H).

Example 68: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Glu)(5Ap)

By following the method of Example 53, 173 mg (82%) of the titlecompound as colorless powder was prepared from 301 mg (0.32 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys[Boc-Pro-Ala-Glu(OBzl)]-OBzl.Mp 194.7-196.1° C.; [α]_(D) ²⁵=−2.3 (c=0.25, CH₃OH); ESI-MS (m/e): 661[M−H]⁻; IR(KBr): 3053.3, 2945.3, 2370.5, 2320.4, 1651.1, 1546.9, 1533.4,1448.5, 1388.8, 1240.2, 1165.0, 1045.4, 644.2; ¹HNMR (300 MHz, D₂O)δ/ppm=6.78 (s, 1H), 6.67 (s, 1H), 4.42-4.28 (m, 5H), 3.29 (m, 2H), 3.13(m, 3H), 2.99 (m, 1H), 2.38 (m, 2H), 2.29 (m, 1H), 1.90 (m, 5H), 1.69(s, 3H), 1.55 (s, 3H), 1.31 (m, 6H).

Comparative Example 1: Preparation of3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(6)

By following the method of Example 4, 620 mg (85%) of the title compoundas light pink solid was prepared from 1.00 g (2.03 mmol)3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys-OBzl. Thiscompound 6 was used as a control group in the following experimentalexamples. ESI-MS(m/e): 363[M−H]⁻.

Experimental Example 1: Evaluating the Thrombolytic Activity ofCompounds 5Aa-p of the Present Invention Via Intravenous Administration

1) Evaluation Method

200-220 g male SD rats were anesthetized with 20% urethane solution (6mL/kg, ip). Anesthetized rats were fixed in a supine position, and theright common carotid artery was separated, clamped at the proximal endwith an arterial clip, and penetrated with a suture at the proximal anddistal ends, respectively. The suture at the distal end is clippedtightly by a hemostatic clamp at the skin/fur. Cannulation was performedat the distal end, the arterial clamp was loosened, and about 1 mlarterial blood was discharged into a 1 mL EP vial. 0.1 mL rat arterialblood was injected into a vertically fixed glass tube (15 mm in length,with an inner diameter of 2.5 mm and an outer diameter of 5.0 mm, sealedwith a rubber stopper at the bottom), into which was immediatelyinserted a thrombus immobilization screw made of stainless steel. Thethrombus immobilization screw, formed by coiling of a stainless steelwire having a diameter of 0.2 mm, had a spiral part of 12 mm in length,including 15 coils each having a diameter of 1.0 mm, and a stem of 7.0mm in length which was connected to the spiral part and had aquestion-mark-like shape. 40 min after the blood coagulated, the rubberstopper at the bottom of the glass tube was removed, the stem of thethrombus immobilization screw was fixed by forceps, and thethrombus-wrapped thrombus immobilization screw was carefully taken outfrom the glass tube and precisely weighted.

A bypass cannula was composed of 3 segments. The middle segment was apolyethylene tube having a length of 60 mm and an inner diameter of 3.5mm. The segments on both ends were similar polyethylene tubes having alength of 100 mm, an inner diameter of 1 mm and an outer diameter of 2mm, one end of which was pulled to form a tip, with an outer diameter of1.0 mm (used in inserting into the rat carotid artery or vein), theother end of which was sheathed by a polyethylene tube having a lengthof 7 mm and an outer diameter of 3.5 mm (thickened, used in insertinginto the polyethylene tube of the middle segment). The inner wall of the3-segment cannula was entirely silylated. The thrombus-wrapped thrombusimmobilization screw was placed into the polyethylene tube of the middlesegment, and both ends of the tube sheath the thickened ends of twopolyethylene tubes. The cannula was filled with a heparin solution innormal saline (50 IU/kg) through the tip end by using an injector andwas ready for use.

The left external jugular vein of the rat was separated and penetratedwith a suture at the proximal and distal ends respectively, and thedistal end was ligated. An incision was carefully made on the exposedleft external jugular vein, and the tip of the bypass cannula preparedas described above was inserted into the proximal end of the incision inthe left external jugular vein, away from the stem of the thrombusimmobilization screw in the middle segment of the bypass cannula (whichaccommodated the accurately weighed thrombus immobilization screw). Aprecise amount of heparin in saline (50 IU/kg) was injected through thetip at the other end by using an injector. At this moment, withoutremoving the injector from the polyethylene tube, the tube between theinjector and the polyethylene tube was clamped with a hemostatic clamp.The blood flow was stopped by clamping the proximal end of the rightcommon carotid artery with an arterial clip, and an incision was cutcarefully across the common carotid artery near the clip. The injectorwas pulled out of the polyethylene tube through the tip, and the tip ofthe polyethylene tube was then inserted into the proximal end of theartery incision. Both ends of the bypass cannula were fixed to theartery or vein with #4 sutures.

A scalp needle was used to pass saline solution, a urokinase in salinesolution or varied concentrations of the compounds in saline solutionthrough the middle section of the bypass cannula (which accommodated theaccurately weighed thrombus immobilization screw) to a position at theproximal vein and away from the thrombus immobilization screw. Theartery clip was then removed to allow blood to flow from the artery tothe vein through the bypass cannula. A rat arteriovenous bypassthrombolysis model was thus established. The solution in the injectorwas slowly injected into blood (about 6 mim), allowing normal saline,urokinase (positive control), or compounds of the present invention toact on the thrombus through blood circulation in the order ofvein-heart-artery. The process was timed at the beginning of injection,and the thrombus immobilization screw was removed from the bypasscannula after 1 hour and accurately weighed. The difference in the massof the thrombus immobilization screw in the rat bypass cannula beforeand after the administration was determined to evaluate the in vivothrombolytic activity of compounds. The weight reduction of the thrombuswas expressed by average values and standard deviation (x±SD).

2) Administration Method and Dose

The administration method is intravenous injection. The blank controlwas normal saline solution, and the dose was 3 mL/kg. The positivecontrol was urokinase, and the dose was 20000 U/kg, which was equal to1.68 mg/kg. The dose of compounds 5Aa-p of the present invention was 0.1nmol/kg.

3) Evaluation Results

The thrombolytic activity was expressed by the weight reduction of thethrombus (x±SD mg), and the results were listed in Table 1. Data showedthat compounds 5Aa-p at a dose of 0.1 nmol/kg via intravenousadministration could effectively lyse the thrombus. (p<0.01 comparedwith normal saline solution). The thrombolytic activities of compounds5Aa, 5Af, 5Ag, 5Ak, and 5Ao were comparable to that of urokinase at adose of 20000 U/kg. Among the compounds with higher thrombolyticactivity, 5Aa, 5Ad, 5Af, 5Ag and 5Ak, compound 5Ak showed the highestthrombolytic activity, thus the dose dependant relationship thereof wasfurther evaluated.

TABLE 1 The thrombolytic activity of compounds 5Aa-p through intravenousinjection. Weight reduction Weight reduction Compounds in thrombusCompounds in thrombus normal saline  9.25 ± 1.59 urokinase 18.95 ±2.73^(a) solution 5Aa 19.35 ± 2.76^(b) 5Ai 16.00 ± 2.03^(a) 5Ab 17.12 ±1.64^(a) 5Aj 15.23 ± 2.45^(a) 5Ac 16.23 ± 2.81^(a) 5Ak 19.38 ± 2.81^(b)5Ad 18.11 ± 2.48^(b) 5Al 14.65 ± 2.86^(a) 5Ae 13.63 ± 1.92^(a) 5Am 16.65± 2.52^(a) 5Af 17.92 ± 1.69^(b) 5An 16.44 ± 2.32^(a) 5Ag 18.98 ±2.14^(b) 5Ao 15.86 ± 2.21^(a) 5Ah 17.10 ± 1.79^(a) 5Ap 16.03 ± 2.14^(a)6 13.87 ± 3.07^(a) PAK 14.17 ± 1.84^(a) 6 + PAK 15.37 ± 3.00^(a) n = 10;^(a)p < 0.01 compared with normal saline solution; ^(b)p < 0.01 comparedwith normal saline solution; p < 0.05 compared with compound 6, PAK andcompound 6 + PAK; p > 0.05 compared with urokinase

Experimental Example 2: Evaluating the Dose-Effect Relationship for theThrombolytic Activity of the Compound 5Ak of the Present Invention ViaIntravenous Administration

Intravenous injection administration was performed according to themethod of example 1. The blank control was normal saline solution, andthe dose was 3 mL/kg. The positive control was urokinase, and the dosewas 20000 U/kg, which was equal to 1.68 mg/kg. The high, medium and lowdoses of the compound 5Ak were 0.1 nmol/kg, 0.01 nmol/kg, and 0.001nmol/kg, respectively. The results are listed in Table 2. Data showedobvious dose-effect relationship of compound 5Ak.

TABLE 2 The dose-effect relationship for thrombolysis of the compound5Ak of the present invention Compounds Weight reduction in thrombus (x ±SD mg) normal saline solution, 10.55 ± 2.52 3 mL/kg Urokinase, 20000U/kg 25.61 ± 3.87 5Ak, 0.1 nmol/kg  21.61 ± 4.62^(a) 5Ak, 0.01 nmol/kg  14.62 ± 2.46 _(b) 5Ak, 0.001 nmol/kg   11.73 ± 2.15 ^(c) n = 10; ^(a)p< 0.01 compared with normal saline solution and 0.01 nmol/kg 5Ak; _(b) p< 0.01 compared with normal saline solution and 0.001 nmol/kg 5Ak; ^(c)p > 0.05 compared with normal saline solution.

Experimental Example 3: Evaluating the Antithrombotic Activity ofCompounds 5Aa-p of the Present Invention Via Intravenous Administration

1) Evaluation Method

A cannula was composed of 3 segments. The middle segment has a length of80 mm and an inner diameter of 3.5 mm. The segments on both ends weresimilar polyethylene tubes having a length of 100 mm, an inner diameterof 1 mm and an outer diameter of 2 mm, one end of which was pulled toform a tip (used in inserting into the rat carotid artery or vein), Theinner wall of the 3-segment cannula was entirely silylated. Thepre-weighed, 60 mm in length thread was placed in the middle segment ofthe polyethylene tube. Both ends of the wider tube sheathed theun-narrowed ends of two polyethylene tubes respectively (0.5 mm of thethread was restrained by one of the segment to fix the thread). Thecannula was filled with a heparin solution in normal saline (50 IU/kg)through the tip end by using an injector and was ready for use.

200-220 g male SD rats were anesthetized with 20% urethane solution (6mL/kg, ip). Anesthetized rats were fixed in a supine position, and theleft external jugular vein was separated and penetrated with a suture atthe proximal and distal ends respectively, and the distal end wasligated. An incision was carefully made on the exposed left externaljugular vein, and the tip end without thread-restraining of the bypasscannula prepared as described above was inserted into the proximal endof the open incision in the left external jugular vein. A precise amountof heparin in normal saline (50 IU/kg) was injected through the tip atthe other end by using an injector. Then, the injector was replaced, andprecise amount of drug was injected by the same way. At this moment,without removing the injector from the ethylene tube, the right commoncarotid artery was separated, and the proximal end was clipped by anartery clip. The right common carotid artery was penetrated with asuture at the proximal and distal ends respectively, and the distal endwas ligated. An incision was carefully cut across the right commoncarotid artery near the artery clip. The injector was pulled out of thepolyethylene tube through the tip, and the tip of the polyethylene tubewas then inserted into the proximal end of the artery incision. Bothends of the bypass cannula were fixed to the artery or vein with #4sutures. The artery clip was then removed to allow blood to flow fromthe artery to the vein through the bypass cannula. A rat arteriovenousbypass thrombolysis model was thus established. The process was timedfrom the beginning of circulation, the thread attached with thrombus wastaken out from the bypass cannula after 15 minutes and accuratelyweighed. The difference in the mass of the thread before and after wasdetermined as the wet weight of the thrombus, which was statisticallyanalyzed and used to evaluate the in vivo antithrombotic activities ofthe compounds. The wet weight of the thrombus was expressed as averagevalue and standard deviation (x±SD).

2) Drug Administration Method and Dose

The drug administration method is intravenous injection. The blankcontrol was normal saline solution, and the dose was 1 mL/kg. Thepositive control was aspirin, and the dose was 9 mg/kg. The dose ofcompounds 5Aa-p of the present invention was 0.1 nmol/kg.

3) Results

The antithrombotic activities were expressed as the wet weight ofthrombus (x±SD), and the results were listed in Table 3. Data showedthat the dose of the compound 5Ak at 0.1 nmol/kg via intravenousadministration may effectively inhibit the formation of thrombus(p<0.001, compared with normal saline solution), and the dose dependantrelationship of 5Ak with higher activity was thus further evaluated.

TABLE 3 The in vivo antithrombotic experiment of compound 5Aa-p of thepresent invention via intraveneus administration wet weight wet weightof thrombus of thrombus Compounds (x ± SD mg) Compounds (x ± SD mg) NS65.40 ± 2.73  Aspirin 46.95 ± 5.21^(a) 5Aa 54.04 ± 6.43^(a) 5Ai 56.12 ±5.55^(a) 5Ab 54.17 ± 6.61^(a) 5Aj 50.56 ± 3.96^(a) 5Ac 52.93 ± 3.96^(a)5Ak 51.93 ± 2.74^(a) 5Ad 51.91 ± 4.97^(a) 5Al 54.20 ± 3.93^(a) 5Ae 57.20± 6.43^(a) 5Am 58.14 ± 2.30^(a) 5Af 54.01 ± 4.25^(a) 5An 55.11 ±4.76^(a) 5Ag 54.28 ± 3.57^(a) 5Ao 48.34 ± 3.29^(a) 5Ah 50.67 ± 3.71^(a)5Ap 49.97 ± 6.32^(a) n = 10; 5Aa-p (i.v.): 0.1 nmol/kg; Aspirin: 9mg/kg; ^(a)p < 0.001 compared with NS.

Experimental Example 4: Evaluating the Dose-Effect Relationship forAntithrombotic Activity of Compound 5Ak of the Present Invention ViaIntravenous Administration

1) Evaluation Method

The method was the same as in example 3.

2) Drug Administration Method and Dose

The drug administration method is intravenous injection. The blankcontrol was normal saline solution, and the dose was 3 mL/kg. Thepositive control was aspirin, and the dose was 9 mg/kg. The high, mediumand low doses of the compound 5Ak were 0.1 nmol/kg, 0.01 nmol/kg, and0.001 nmol/kg respectively.

3) Results

The results were listed in Table 4, and compound 5Ak showed obviousdose-effect relationship.

TABLE 4 In vivo dose-effect realtionship for antithrombotic activity ofcompound 5Ak of the present invention Compounds wet weight of thrombus(x ± SD mg) NS 63.46 ± 3.67  Aspirin 45.62 ± 2.60  5Ak, 0.1 nmol/kg52.16 ± 2.02^(a) 5Ak, 0.01 nmol/kg 57.34 ± 3.73^(b) 5Ak, 0.001 nmol/kg61.91 ± 3.27^(c) n = 10; ^(a)p<0.01 compared with normal saline solutionand 0.01 nmol/kg 5Ak; ^(b)p < 0.01 compared with normal saline solutionand 0.001 nmol/kg 5Ak; ^(c)p > 0.05 compared with normal salinesolution.

Experimental Example 5: Evaluating the Therapeutic Effect of theCompound 5Ak of the Present Invention in Stroke Rats

For evaluating the therapeutic effect of compounds 5Aa-p in stroke rats,compound 5Ak was selected as a representative and its therapeutic effectin stroke rats was evaluated by the method below.

1) Evaluation Method

SD male rats (280-300 g) were randomly divided to UK positive controlgroup at a dose of 20000 IU/kg, blank control group with normal salinesolution, compound control group with PAK at a dose of 5 μmol/kg, andcompound 5Ak group at a dose of 1 μmol/kg (high), 0.1 μmol/kg (medium),and 0.01 μmol/kg (low). A 10% chloral hydrate solution (400 mg/kg) wasinjected intraperitoneally into rats for anesthesia. A vertical incisionof about 2 cm in length was made on the right side near the center ofthe neck, and the right common carotid artery (CCA), external carotidartery (ECA) and internal carotid artery (ICA) were separated along themargin of the inner side of sternocleidomastoid muscles. The incision atthe internal carotid artery and the proximal end of the common carotidartery were clipped respectively with noninvasive arterial clips. Asmall incision was made on the external carotid artery, and the distalend of the external carotid artery was ligated. The arterial clip at theproximal end of the common carotid artery was released, and 10 μl bloodwas drawn. After blood was drawn, the proximal end of the common carotidartery was again clipped with a noninvasive arterial clip. The 10 μlblood drawn was placed in a 1 mL EP vial and kept at RT for 30 min toallow the coagulation of blood and then transferred into a −20° C.refrigerator for 1 hour to allow the formation of solid coagulation. 1hour later, the blood clots were taken out, 1 mL normal saline was addedtherein, and then the blood clots were broken into relatively uniformmicrothrombus by a steel spatula. The microthrombus suspension was thentransferred into a 1 mL injector for use. When the clip on the internalcarotid artery of the rat was released, the 1 mL thrombus suspension inthe injector was slowly injected from the external carotid artery of therat to its proximal end, and then was injected into the brain of the ratthrough the internal carotid artery. Subsequently, the proximal end ofthe external carotid artery was ligated, the arterial clips on theinternal carotid artery and the common carotid artery were released, andblood flow was restored. The common jugular vein was separated, and theninjected with UK at the dose of 20000 IU/kg, normal saline solution, orcompound 5Ak at the dose of 1 μmol/kg. The vein was ligated. 3 drops ofpenicillin was dropped on the wound. The wound was sewed, and animalswere waited for awake.

24 hours after the rats were awake, the degree of damage in neuralfunction was evaluated by the Zealonga method. A score of 0 indicated nosign of loss in neural function, 1 indicated the front limbs on theundamaged side could not stretch out, 2 indicated walking toward theundamaged side, 3 indicated tail-chasing walking in circles toward theundamaged side, 4 indicated involuntary walking with disturbance ofconsciousness, and 5 indicated death. The evaluation results werestatistically analyzed and subjected to t-test.

After the rats were awake for 24 hours and assessed for their degree ofdamage in neural function by Zealonga method, they were anesthetizedwith urethane followed by immediate decapitation and removal of thebrain. Brain tissues were kept in a −20° C. refrigerator for 2 hours,and coronal sections of about 2 mm were successively sliced from theprefrontal end for a total of 5 sections and then placed into a 2% TTCsolution to incubate without light at 37° C. for 30 min. The colorchange in brain sections was observed: normal brain tissues were stainedred by TTC, while ischemic brain tissues appeared in white. Photographswere taken by using a digital camera and processed with image statisticssoftware, and the volume of infarction in brain tissues and the area ofnormal brain tissues in the coronal sections were calculated. The ratioof the cerebral infarction volume of each group was statisticallycalculated and subject to t-test.

2) Evaluation Result

The evaluated data according to the Zealong method after the rats awakewere listed in Table 5. The percentages of cerebral infarction volumewere listed in Table 6.

TABLE 5 Evaluation by the Zealong method after the rats awake for 24hours Compounds Neural function scores (x ± SD) normal saline solution2.90 ± 0.99  urokinase, 20000 IU/kg 1.30 ± 0.95^(a) 5Ak, 1 μmol/kg 1.10± 0.74^(a) 5Ak, 0.1 μmol/kg 1.40 ± 1.07^(b) 5Ak, 0.01 μmol/kg 2.30 ±0.95^(c) PAK, 5 μmol/kg 2.20 ± 1.60  n = 9; ^(a)p < 0.05 compared withnormal saline solution and 0.1 μmol/kg 5Ak; ^(b)p < 0.05 compared withnormal saline solution and 0.01 μmol/kg 5Ak; ^(c)p > 0.05 compared withnormal saline solution and 5 μmol/kg PAK

TABLE 6 Percentage of cerebral infarction volume in rats CompoundsPercentage of infarction volume (x ± SD %) normal saline solution 22.99± 5.08 urokinase, 20000 IU/kg  4.60 ± 2.09 5Ak, 1 μmol/kg  3.44 ±1.99^(a) 5Ak, 0.1 μmol/kg   9.88 ± 2.52^(b) 5Ak, 0.01 μmol/kg  18.05 ±5.77^(c) PAK, 5 μmol/kg 18.36 ± 7.38 n = 7; ^(a)p < 0.01 compared withnormal saline solution, p < 0.05 compared with 0.1 μmol/kg 5Ak, p > 0.05compared with urokinase; ^(b)p < 0.05 compared with normal salinesolution and 0.1 μmol/kg 5Ak; ^(c)p > 0.05 compared with normal salinesolution.

The data shown in Tables 5 and 6 suggested that compound 5Ak mayeffectively prevent the brain ischemia rats from occurrence ofdyskinesia and cerebral infarction. This function of compound 5Ak wasdose dependent.

Experimental Example 6: Evaluating the Free Radical Scavenging Activityof Compounds 5Aa-p of the Present Invention

1) Hydroxyl Free Radical Scavenging Activity

11.316 mg DMPO (dimethylpyridine-N-oxide, Sigma) was dissolved in 1 mLof pure water to obtain 0.1 M DMPO solution. 2.78 g FeSO₄.7H₂O wasdissolved in 1 mL of pure water to obtain 10 mM solution. 30% medicalH₂O₂ was diluted to 0.2%.

The first peak height of the OH signal of 2.5 μL FeSO₄.7H₂O solution+2.5μL DMPO solution+5 μL H₂O₂ solution+5 μL water was measured and repeatedfor 6 times. This peak height defined as the known peak height of OHsignal. The first peak height of the OH signal of 2.5 μL FeSO₄.7H₂Osolution+2.5 μL DMPO solution+5 μL H₂O₂ solution+5 μL solution of one ofthe compounds 5Aa-p was measured and repeated for 6 times. This peakheight defined as the residual peak height of the OH signal scavenged byone of the compounds 5Aa-p.

Scavenging ratio=(known peak height of OH signal−residual peak height ofOH signal of compounds 5Aa-p)/known peak height of OH signal

2) NO Radical Scavenging Activity

7.325 mg MGD (N-methyl-glucamine dithiocarbamate, Sigma) was dissolvedin 1 mL of pure water to obtain 25 mM MGD solution. 3.475 g FeSO₄.7H₂Owas dissolved in 1 mL of pure water to obtain 12.5 mM solution. 25 mgSNAP (S-Nitroso-acetyl penicillamine) was dissolved in 1 mL of purewater to obtain 110 μM green mother liquid, which was diluted 100 foldsto obtain 1 μM SNAP solution.

The first peak height of NO signal of 5 μL MGD solution+5 μL FeSO₄.7H₂Osolution+5 μL SNAP solution+5 μL water was measured and repeated for 6times. This peak height defined as the known peak height of NO signal.The first peak height of NO signal of 5 μL MGD solution+5 μL FeSO₄.7H₂Osolution+5 μL SNAP solution+5 μL solution of one of the compounds 5Aa-pwas measured and repeated for 6 times. This peak height defined as theresidual peak height of the NO signal scavenged by one of the compounds5Aa-p.

Scavenging ratio=(known peak height of NO signal−residual peak height ofNO signal of compounds 5Aa-p)/known peak height of NO signal

3) Superoxide Anionic Radical Scavenging Activity

0.3 g xanthine was dissolved in 1 mL of pure water to obtain 0.5 Mxanthine solution (milk white color, largely insoluble). Commerciallyavailable raw solution of xanthine oxidase was diluted 10 folds toobtain solution of xanthine oxidase. Saturated DETAPAC solution wasdiluted 20 folds to obtain 0.9 mM solution. 11.316 mg DMPO was dissolvedin 1 mL of pure water to obtain 0.1 M DMPO solution.

5 μL DMPO solution+5 μL DETAPAC solution+5 μL xanthine solution+5 μLxanthine oxidase solution+5 μL compounds 5Aa-p solution

The first peak height of superoxide anion signal of 5 μL DMPO solution+5μL DETAPAC solution+5 μL xanthine solution+5 μL xanthine oxidasesolution+5 μL water was measured and repeated for 6 times. This peakheight defined as the known peak height of superoxide anion signal. Thefirst peak height of superoxide anion signal of 5 μL DMPO solution+5 μLDETAPAC solution+5 μL xanthine solution+5 μL xanthine oxidase solution+5μL solution of one of compounds 5Aa-p was measured and repeated for 6times. This peak height defined as the residual peak height of thesuperoxide anion signal scavenged by one of the compounds 5Aa-p.

Scavenging ratio=(known peak height of superoxide anion signal−residualpeak height of superoxide anion signal of compounds 5Aa-p)/known peakheight of superoxide anion signal

Results were listed in Table 7. Data showed that the EC₅₀ of thecompounds 5Aa-p for three free radical scavenging activity was 0.4-0.7mM. Compounds 5Aa-p have obvious free radical scavenging activity.

TABLE 7 EC₅₀ of the compounds 5Aa-p for free radical scavenging activityEC₅₀ of scavenging the free radicals below (x ± SD × 10⁻⁴ M) Comopounds•NO •OH Superoxide anionic radical 5Aa 5.99 ± 0.38 4.59 ± 0.66 4.77 ±0.56 5Ab 5.32 ± 0.73 4.71 ± 0.71 4.71 ± 0.34 5Ac 4.63 ± 0.92 3.78 ± 0.853.66 ± 0.71 5Ad 4.53 ± 0.41 3.69 ± 0.32 3.54 ± 0.71 5Ae 6.17 ± 0.88 4.66± 0.47 4.81 ± 0.45 5Af 5.68 ± 0.95 5.44 ± 0.43 4.85 ± 0.31 5Ag 6.09 ±0.95 4.22 ± 0.61 4.67 ± 0.94 5Ah 5.23 ± 0.85 4.13 ± 0.73 4.01 ± 0.72 5Ai5.27 ± 0.90 4.95 ± 0.61 4.52 ± 0.64 5Aj 5.10 ± 0.75 4.16 ± 0.28 4.17 ±0.95 5Ak 4.99 ± 0.89 4.22 ± 0.82 3.63 ± 0.94 5Al 5.03 ± 0.41 4.89 ± 0.324.14 ± 0.97 5Am 6.47 ± 0.88 4.66 ± 0.77 3.71 ± 0.45 5An 6.59 ± 0.89 5.72± 0.72 3.83 ± 0.94 5Ao 4.73 ± 0.99 3.96 ± 0.48 4.35 ± 0.65 5Ap 4.77 ±0.91  4.0 ± 0.51 4.20 ± 0.73 n = 6, EC₅₀ = X ± SD; compoundconcentration was X × 10⁻⁴ M

Experimental Example 7: TEM Observations of the Compound 5Aa-p of thePresent Invention

Samples were prepared as 1×10⁻⁷ M, 1×10⁻⁹ M, 1×10¹¹ M solution bydistillated water, respectively. A small amount (about 10 μl) of thesolution was taken and dropped onto the surface of a copper grid with afilter paper placed underneath, for air dry. The morphology and particlesize were then observed under transmission electron microscope (JEOL,JEM-1230) and recorded in photographs.

In aqueous solution, the compounds 5Aa-p may be self-assembled tonanospherical structure having a diameter between 20-210 nm, wherein thediameter was mostly between 20-100 nm. These nanospheres were coupled tovarious shapes of nanonet or nano necklase, etc. For example, when thein vivo concentration was 1×10⁻⁹ M (theoritical drug concentration inblood), the TEM photographs were respectively shown (FIG. 9). In FIG. 9,the compounds 5Aa-p were respectively corresponding to figure number 5a,5b, 5c, 5d, 5e, 5f, 5g, 5h, 5i, 5j, 5k, 5l, 5m, 5n, 5o, and 5p.

Experimental Example 8: Experiment of Successive Treatment with 1μMol/Kg 5Ak for 6 Times in Stroke Rats after 4 Hours from the StrokeOnset

The therapeutic effect was evaluated by neurological functional scores.The lower the score was, the better the therapeutic effect was. A 10%chloral hydrate solution was injected intraperitoneally into male SDrats at a dosage of 400 mg/kg body weight for anesthesia. A longitudinalincision was made at the center of the neck, and the right commoncarotid artery trunk was separated (about 3 cm in length). Externalcarotid artery branches were each separated and ligated at the hyoidlevel, and the internal carotid artery was separated from the swollenpart of the neck. The incision of the internal carotid artery and theproximal end of the common carotid artery were clipped respectively withnoninvasive arterial clips, and the distal end of the external carotidartery was ligated. A catheter containing 0.5 mL thrombus suspension innormal saline was inserted in the external carotid artery trunk. Whenthe clip on the internal carotid artery was released, the 0.5 mLthrombus suspension in normal saline in the catheter slowly flew fromthe external carotid artery to its proximal end, and then was injectedinto the arteries in brain through the internal carotid artery.Subsequently, the proximal end of the internal carotid artery wasligated, the arterial clips on the internal carotid artery and thecommon carotid artery were released, and blood flow was restored. Afterthe wound was stitched up, 20,000 IU penicillin was intramuscularlyinjected for prevention from infection. 4 hours later, compound 5Ak innormal saline (at the dose of 1 μmol/kg, n=10) was infused forconsecutive 6 days and observed for 7 days. Self-comparison wasperformed each day, and the neurological deficit degree was evaluated bythe Zealonga method. A score of 0 indicated no sign of loss in neuralfunction, 1 indicated the front limbs on the undamaged side could notstretch out, 2 indicated walking toward the undamaged side, 3 indicatedtail-chasing walking in circles toward the undamaged side, 4 indicatedinvoluntary walking with disturbance of consciousness, and 5 indicateddeath. The evaluation results were listed in Table 8. Data showed thatin rats receiving a successive 6-day treatment with 1 μmol/kg 5Ak onceper day after 4 hours from stroke onset, no death occurred. Eight of theten rats improved to no neurological deficits. Only 2 rats had slightneurological deficits. Therefore, the dose of 1 μmol/kg 5Ak has definitetherapeutic effect after 4 hours from stroke onset.

TABLE 8 Therapeutic effect of rats receiving treatment of 1 μmol/kg 5Akafter 4 hours from stroke onset Daily neural function scores (Mean ± SD)Evaluation and number of rats under each score time 0 score 1 score 2scores 3 scores 4 scores 5 scores 1^(st) day 3 5 1 1 0 0 2^(nd) day 5 41 0 0 0 3^(rd) day 3 6 1 0 0 0 4^(th) day 8 1 1 0 0 0 5^(th) day 6 4 0 00 0 6^(th) day 8 2 0 0 0 0

Experimental Example 9: Experiment of Successive Treatment with 1μMol/Kg 5Ak for 6 Times in Stroke Rats after 6 Hours from the StrokeOnset

The therapeutic effect was evaluated by neurological functional scores.The lower the score was, the better the therapeutic effect was. A 10%chloral hydrate solution was injected intraperitoneally into male SDrats at a dosage of 400 mg/kg of body weight for anesthesia. Alongitudinal open incision was made at the center of the neck, and theright common carotid artery trunk was separated (about 3 cm in length).External carotid artery branches were each separated and ligated at thehyoid level, and the internal carotid artery was separated at theswollen part of the neck. The incision in the internal carotid arteryand the proximal end of the common carotid artery were clippedrespectively with noninvasive arterial clips, and the distal end of theexternal carotid artery was ligated. A catheter containing 0.5 mLthrombus suspension in normal saline was inserted in the externalcarotid artery trunk. When the clip on the internal carotid artery wasreleased, the 0.5 mL thrombus suspension in normal saline in thecatheter slowly flew from the external carotid artery to its proximalend, and then was injected into the arteries in brain through theinternal carotid artery. Subsequently, the proximal end of the internalcarotid artery was ligated, the arterial clips on the internal carotidartery and the common carotid artery were released, and blood flow wasrestored. After the wound was stitched up, 20,000 IU penicillin wasintramuscularly injected for prevention from infection. After 6 hours,compound 5Ak in normal saline (at the dose of 1 μmol/kg, n=10) wasinfused consecutive for 6 days and observed for 7 days. Self-comparisonwas performed each day, and the neurological deficit degree wasevaluated by the Zealonga method. A score of 0 indicated no sign of lossin neural function, 1 indicated the front limbs on the undamaged sidecould not stretch out, 2 indicated walking toward the undamaged side, 3indicated tail-chasing walking in circles toward the undamaged side, 4indicated involuntary walking with disturbance of consciousness, and 5indicated death. The evaluation results were listed in Table 9. Datashowed that in rats receiving a successive 6-day treatment with 1μmol/kg 5Ak once per day after 6 hours from stroke onset, only one ratdied accidentally. Six of the nine rats recovered to no neurologicaldeficits. Only 3 rats had slight neurological deficits. Therefore, thedose of 1 μmol/kg 5Ak has definite therapeutic effect after 6 hours fromstroke onset.

TABLE 9 Therapeutic effect of rats receiving treatment of 1 μmol/kg 5Akafter 6 hours from stroke onset Daily neural function scores (Mean ± SD)Evaluation and Number of rats under each score time 0 score 1 score 2scores 3 scores 4 scores 5 scores 1^(st) day 0 4 4 2 0 0 2^(nd) day 0 72 1 0 0 3^(rd) day 1 6 2 1 0 0 4^(th) day 4 4 1 0 0 1 5^(th) day 7 2 0 00 0 6^(th) day 6 3 0 0 0 0

Experimental Example 10: Experiment of Successive Treatment with 1μMol/Kg 5Ak for 6 Times in Stroke Rats after 24 Hours from the StrokeOnset

The therapeutic effect was evaluated by neurological functional scores.The lower the score was, the better the therapeutic effect was. A 10%chloral hydrate solution was injected intraperitoneally into male SDrats at a dosage of 400 mg/kg of body weight for anesthesia. Alongitudinal incision was made at the center of the neck, and the rightcommon carotid artery trunk was separated (about 3 cm in length).External carotid artery branches were each separated and ligated at thehyoid level, and the internal carotid artery was dissected at theswollen part of the neck. The incisions in the internal carotid arteryand the proximal end of the common carotid artery were clippedrespectively with noninvasive arterial clips, and the distal end of theexternal carotid artery was ligated. A catheter containing 0.5 mLthrombus suspension in normal saline was inserted in the externalcarotid artery trunk. When the clip on the internal carotid artery wasreleased, the 0.5 mL thrombus suspension in normal saline in thecatheter slowly flew from the external carotid artery to its proximalend, and then was injected into the arteries in brain through theinternal carotid artery. Subsequently, the proximal end of the internalcarotid artery was ligated, the arterial clips on the internal carotidartery and the common carotid artery were released, and blood flow wasrestored. After the wound was stitched up, 20,000 IU penicillin wasintramuscularly injected for prevention from infection. After 24 hours,compound 5Ak in normal saline (at the dose of 1 μmol/kg, n=10) wasinfused consecutive for 6 days and observed for 7 days. Self-comparisonwas performed each day, and the neurological deficit degree wasevaluated by the Zealonga method. A score of 0 indicated no sign of lossin neural function, 1 indicated the front limbs on the undamaged sidecould not stretch out, 2 indicated walking toward the undamaged side, 3indicated tail-chasing walking in circles toward the undamaged side, 4indicated involuntary walking with disturbance of consciousness, and 5indicated death. The evaluation results were listed in Table 10. Datasuggested that 2 rats died after 24 hours from stroke onset. In the rest8 rats receiving a 6-day successive treatment by using 1 μmol/kg 5Akonce per day, no death occurred. Three of the 8 rats recovered to noneurological deficits. Four rats had slight neurological deficits, andone rat had obvious neurological deficits. Therefore, the dose of 1μmol/kg 5Ak still has definite therapeutic effect after 24 hours fromstroke onset.

TABLE 10 Therapeutic effect of rats receiving treatment of 1 μmol/kg 5Akafter 24 hours from stroke onset Daily neural function scores (Mean ±SD) Evaluation and number of rats under each score time 0 score 1 score2 scores 3 scores 4 scores 5 scores 1^(st) day 1 4 0 2 1 0 2^(nd) day 23 3 1 0 0 3^(rd) day 3 2 3 0 0 0 4^(th) day 2 3 3 0 0 0 5^(th) day 4 3 10 0 0 6^(th) day 3 4 1 0 0 0

Experimental Example 11: Experiment of Successive Treatment with 2.5μMol/Kg 5Ak for 6 Times in Stroke Rats after 6 Hours from the StrokeOnset

The therapeutic effect was evaluated by neurological functional scores.The lower the score was, the better the therapeutic effect was. A 10%chloral hydrate solution was injected intraperitoneally into male SDrats at a dosage of 400 mg/kg of body weight for anesthesia. Alongitudinal incision was made at the center of the neck, and the rightcommon carotid artery trunk was separated (about 3 cm in length).External carotid artery branches were each separated and ligated at thehyoid level, and the internal carotid artery was separated at theswollen part of the neck. The incision at the internal carotid arteryand the proximal end of the common carotid artery were clippedrespectively with noninvasive arterial clips, and the distal end of theexternal carotid artery was ligated. A catheter containing 0.5 mLthrombus suspension in normal saline was inserted in the externalcarotid artery trunk. At the same time when the clip on the internalcarotid artery was released, the 0.5 mL thrombus suspension in normalsaline in the catheter slowly flew from the external carotid artery toits proximal end, and then was injected into the arteries in brainthrough the internal carotid artery. Subsequently, the proximal end ofthe internal carotid artery was ligated, the arterial clips on theinternal carotid artery and the common carotid artery were released, andblood flow was restored. After the wound was stitched up, 20,000 IUpenicillin was intramuscularly injected for prevention from infection.After 6 hours, compound 5Ak in normal saline (at the dose of 2.5μmol/kg, n=10) was infused consecutive for 6 days and observed for 7days. Self-comparison was performed each day, and the neurologicaldeficit degree was evaluated by the Zealonga method. A score of 0indicated no sign of loss in neural function, 1 indicated the frontlimbs on the undamaged side could not stretch out, 2 indicated walkingtoward the undamaged side, 3 indicated tail-chasing walking in circlestoward the undamaged side, 4 indicated involuntary walking withdisturbance of consciousness, and 5 indicated death. The evaluationresults were listed in Table 11. Data showed that in nine rats receivinga successive 6-day treatment with 2.5 μmol/kg 5Ak once per day after 6hours from stroke onset, no death occurred. Seven of the nine ratsimproved to no neurological deficits. One rats had slight neurologicaldeficits. Therefore, the dose of 2.5 μmol/kg 5Ak obviously has bettertherapeutic effect than 1 μmol/kg 5Ak after 6 hours from stroke onset.

TABLE 11 Therapeutic effect of rats receiving treatment of 2.5 μmol/kg5Ak after 6 hours from stroke onset Daily neural function scores (Mean ±SD) Evaluation and number of rats under each score time 0 score 1 score2 scores 3 scores 4 scores 5 scores 1^(st) day 1 7 0 1 0 0 2^(nd) day 34 2 0 0 0 3^(rd) day 6 2 1 0 0 0 4^(th) day 6 3 0 0 0 0 5^(th) day 5 4 00 0 0 6^(th) day 7 2 0 0 0 0

Experimental Example 12: Experiment of Successive Treatment with 2.5μMol/Kg 5Ak for 6 Times in Stroke Rats after 24 Hours from the StrokeOnset

The therapeutic effect was evaluated by neurological functional scores.The lower the score was, the better the therapeutic effect was. A 10%chloral hydrate solution was injected intraperitoneally into male SDrats at a dosage of 400 mg/kg of body weight for anesthesia. Alongitudinal incision was made at the center of the neck, and the rightcommon carotid artery trunk was separated (about 3 cm in length).External carotid artery branches were each separated and ligated at thehyoid level, and the internal carotid artery was separated at theswollen part of the neck. The incision in the internal carotid arteryand the proximal end of the common carotid artery were clippedrespectively with noninvasive arterial clips, and the distal end of theexternal carotid artery was ligated. A catheter containing 0.5 mLthrombus suspension in normal saline was inserted in the externalcarotid artery trunk. When the clip on the internal carotid artery wasreleased, the 0.5 mL thrombus suspension in normal saline in thecatheter slowly flew from the external carotid artery to its proximalend, and then was injected into the arteries in brain through theinternal carotid artery. Subsequently, the proximal end of the internalcarotid artery was ligated, the arterial clips on the internal carotidartery and the common carotid artery were released, and blood flow wasrestored. After the wound was stitched up, 20,000 IU penicillin wasintramuscularly injected for prevention from infection. After 24 hours,compound 5Ak in normal saline (dose 2.5 μmol/kg, n=10) was infusedconsecutive for 6 days and observed for 7 days. Self-comparison wasperformed each day, and the neurological deficit degree was evaluated bythe Zealonga method. A score of 0 indicated no sign of loss in neuralfunction, 1 indicated the front limbs on the undamaged side could notstretch out, 2 indicated walking toward the undamaged side, 3 indicatedtail-chasing walking in circles toward the undamaged side, 4 indicatedinvoluntary walking with disturbance of consciousness, and 5 indicateddeath. The evaluation results were listed in Table 12. The data showthat 3 rats died after 24 hours from stroke onset. In the rest sevenrats receiving a successive 6-day treatment with 2.5 μmol/kg 5Ak onceper day, two rats died. Three rats recovered to no neurologicaldeficits. One rat had slight neurological deficits, and one rat hadobvious neurological deficits. Therefore, the dose of 2.5 μmol/kg 5Akstill has certain therapeutic effect after 24 hours from stroke onset.

TABLE 12 Therapeutic effect of rats received treatment of 2.5 μmol/kg5Ak after 24 hours from stroke onset Daily neural function scores (Mean± SD) Evaluation and Number of rats under each score time 0 score 1score 2 scores 3 scores 4 scores 5 scores 1^(st) day 1 4 1 0 1 0 2^(nd)day 3 2 1 1 0 0 3^(rd) day 3 2 1 0 0 1 4^(th) day 2 2 1 1 0 0 5^(th) day4 0 1 0 0 1 6^(th) day 3 1 1 0 0 0

Experimental Example 13: Experiment of Successive Treatment with 5μMol/Kg 5Ak for 6 Times in Stroke Rats after 24 Hours from the StrokeOnset

The therapeutic effect was evaluated by neurological functional scores.The lower the score was, the better the therapeutic effect was. A 10%chloral hydrate solution was injected intraperitoneally into male SDrats at a dosage of 400 mg/kg of body weight for anesthesia. Alongitudinal incision was made at the center of the neck, and the rightcommon carotid artery trunk was separated (about 3 cm in length).External carotid artery branches were each separated and ligated at thehyoid level, and the internal carotid artery was separated at theswollen part of the neck. The incision in the internal carotid arteryand the proximal end of the common carotid artery were clippedrespectively with noninvasive arterial clips, and the distal end of theexternal carotid artery was ligated. A catheter containing 0.5 mLthrombus suspension in normal saline was inserted in the externalcarotid artery trunk. When the clip on the internal carotid artery wasreleased, the 0.5 mL thrombus suspension in normal saline in thecatheter slowly flew from the external carotid artery to its proximalend, and then was injected into the arteries in brain through theinternal carotid artery. Subsequently, the proximal end of the internalcarotid artery was ligated, the arterial clips on the internal carotidartery and the common carotid artery were released, and blood flow wasrestored. After the wound was stitched up, 20,000 IU penicillin wasintramuscularly injected for prevention from infection. After 24 hours,compound 5Ak in normal saline (at the dose of 5 μmol/kg, n=10) wasinfused consecutive for 6 days and observed for 7 days. Self-comparisonwas performed each day, and the degree of damage of neurological deficitdegree was evaluated by the Zealonga method. A score of 0 indicated nosign of loss in neural function, 1 indicated the front limbs on theundamaged side could not stretch out, 2 indicated walking toward theundamaged side, 3 indicated tail-chasing walking in circles toward theundamaged side, 4 indicated involuntary walking with disturbance ofconsciousness, and 5 indicated death. The evaluation results were listedin Table 13. The data show that one rat died after 24 hours from strokeonset. In the rest ten rats receiving a successive 6-day treatment with5 μmol/kg 5Ak once per day, no death occurred. Seven rats recovered tono neurological deficits. Three rats had slight neurological deficits.Therefore, the dose of 5 μmol/kg 5Ak has definite therapeutic effectafter 24 hours from stroke onset, and obviously better than the dose of2.5 μmol/kg.

TABLE 13 Therapeutic effect of rats receiving treatment of 5 μmol/kg 5Akafter 24 hours from stroke onset Daily neural function scores (Mean ±SD) Evaluation and number of rats under each score time 0 score 1 score2 scores 3 scores 4 scores 5 scores 1^(st) day 0 5 4 0 1 0 2^(nd) day 26 2 0 0 0 3^(rd) day 2 6 2 0 0 0 4^(th) day 5 5 0 0 0 0 5^(th) day 6 4 00 0 0 6^(th) day 7 3 0 0 0 0

According to the experimental results, the compounds of the presentinvention may form nanostructure to achieve the functions of crossingthe blood-brain barrier. In addition to thrombolytic and antithromboticfunctions, these compounds may also effectively scavenge OH, NO,superoxide anionic and other free radical. Moreover, only low dose wasneeded for effective thrombolysis. A higher dose may demonstrateexcellent effect in treating stroke beyond 4 hours from stroke onset.Therefore, these compounds have good prospects for clinical application.

What is claimed is:
 1. A method for preparing a compound having formulaI:

wherein R₁ and R₂ are independently C₁₋₄ alkyl, T is a linking armhaving an amino group and a second group for linking, and Q is a peptidehaving thrombolytic activity; the method comprises the steps of: (a)coupling the carboxyl group of a compound having formula II with theamino group of the linking arm T to form a compound having formula IM-1:

(b) coupling a terminal group of the peptide Q with the second group ofthe linking arm T in the compound having formula IM-1, to form thecompound having the formula I.
 2. The method of claim 1, wherein thesecond group of the linking arm T is a carboxyl group or an amino group.3. The method of claim 1, wherein the linking arm T is L-Lys, L-Asp, orL-Glu.
 4. The method of claim 1, wherein Q is selected from the groupconsisting of an oligopeptide having a PA (Pro-Ala) sequence, a PAK(Pro-Ala-Lys) sequence, an AKP (Ala-Lys-Pro) sequence, and a KAP(Lys-Ala-Pro) sequence.
 5. The method of claim 1, wherein Q has repeatedunits selected from the group consisting of PAK sequence, AKP sequence,and KAP sequence.
 6. The method of claim 1, wherein Q is a tripeptidehaving formula Q1 or Q2:Pro-Ala-AA  (Q1)AA-Ala-Pro  (Q2) wherein AA is selected from the group consisting ofL-Ala, L-Val, L-Trp, L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr,L-Lys, L-Leu, L-Gln, L-Asn, L-Asp, and L-Glu.
 7. The method of claim 1,wherein R₁ and R₂ both are methyl groups.
 8. The method of claim 1,wherein the formula (I) compound has the following formula Ia, Ib, Ic,Id, Ie, If, Ig, or Ih:

wherein AA is selected from a group consisting of L-Ala, L-Val, L-Trp,L-Tyr, L-Pro, L-Phe, Gly, L-Ser, L-Ile, L-Thr, L-Lys, L-Leu, L-Gln,L-Asn, L-Asp, and L-Glu.
 9. The method of claim 8, wherein AA is L-Lys.10. The method of claim 8, wherein the formula (I) compound has theformula Ia.
 11. The method of claim 10, wherein AA is L-Lys.